Method of treating cancer including administering a human chimeric antibody specific for the ganglioside GD3

ABSTRACT

A humanized chimera antibody, a pharmaceutical composition comprising a humanized chimera antibody and a pharmaceutically acceptable carrier, and a method of treating cancer which comprises administering to a patient a pharmaceutically acceptable amount of the humanized chimera antibody, are disclosed.

This application is a division of application Ser. No. 09/225,322, filedJan. 5, 1999 (now U.S. Pat. No. 6,437,098), which was a division ofapplication Ser. No. 08/454,680 filed May 31, 1995 (now U.S. Pat. No.5,866,692), which was a division of application Ser. No. 08/408,133filed Mar. 21, 1995 (now U.S. Pat. No. 5,750,078), which was acontinuation of application Ser. No. 08/292,178 filed Aug. 17, 1994, nowABN, which was a continuation of application Ser. No. 07/947,674 filedSep. 17, 1992 now ABN, the entire content of which is herebyincorporated by reference in this application.

FIELD OF THE INVENTION

This invention relates to a process for the production of humanizedchimera antibody. In contrast to mouse monoclonal antibody, humanizedchimera antibody does not cause formation of anti-mouse immunoglobulinantibody in the body of a patient. Thus, side effects are reduced oreliminated and half life in blood increases when the chimera antibody isused. Therapeutic effects which are superior to those obtained in thecase of using mouse monoclonal antibody can be obtained in the treatmentof human cancers and the like.

BACKGROUND OF THE INVENTION

It is known that, when mouse antibodies are administered to humans, theyare recognized as foreign substances and cause formation of anti-mouseimmunoglobulin antibodies in the human body, and the thus formedantibodies react with the administered mouse antibodies. As a result,side effects occur (J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349–1363(1985); J. Natl. Cancer Inst., 80, 932 (1988); Proc. Natl. Acad. Sci.U.S.A., 82, 1242 (1985)), the antibodies are cleared away quickly (J.Nucl. Med., 26, 1011 (1985); Blood, 65, 1349–1363 (1985); J. Natl.Cancer Inst., 80, 937 (1988)) and effects of the antibodies are reduced(J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 (1986)). Whenmouse monoclonal antibody is converted into humanized chimera antibody,human anti-mouse, immunoglobulin antibody form in minimal amounts if atall, and the half life of the chimera antibody in human blood is sixtimes as long as that of mouse monoclonal antibody (Proc. Natl. Acad.Sci. U.S.A., 86, 4220 (1989)). In addition, it is probable that the Fcregion of mouse antibody does not fully activate human complement andhuman effector cells, in comparison with the Fc region of humanantibody. For example, the antitumor activity of mouse monoclonalantibody to ganglioside GD₂, which is effected via human effector cells,is improved when the monoclonal antibody is converted into chimeraantibody that has the human antibody Fc region (J. Immunol., 144,1382–1386 (1990)).

Ganglioside is one of the animal cell membrane-constituting glycolipidsand is composed of a sugar chain as a hydrophilic side chain,sphingosine as a hydrophobic side chain and fatty acids. It is knownthat expression of ganglioside varies depending on the type of cells,organs and animal species. In addition, it has been revealed thatquantity and quality of the expressed ganglioside change during thecanceration process of cells (Cancer Res., 45, 2405 (1985)). Forexample, it has been resorted that gangliosides GD₂, GD₃, GM₂ and thelike which hardly exist in normal cells were found in the cells ofneuroblastoma, lung small cell carcinoma and melanoma belonging toneuroectodermal-origin tumor which is said to be highly malignant (J.Exp. Med., 155, 1133 (1982); J. Biol. Chem., 257, 12752 (1982); CancerRes., 47, 225 (1987); ibid., 47, 1098 (1987); ibid., 45, 2642 (1985);Proc. Natl. Acad. Sci. U.S.A., 80, 5392 (1983)).

Ganglioside GD₃ has been found most frequently in melanoma cells amongthe neuroectodermal-origin tumors, and anti-ganglioside GD₃ monoclonalantibodies (to be referred to as “anti-GD₃ monoclonal antibody”hereinafter) belonging to the mouse IgM class and IgG class have beenreported (Int. J. Cancer, 29, 269 (1982); J. Biol. Chem., 257, 12752(1982); Cancer Res., 47, 225 (1987); Acta Neuropathol., 79, 317 (1989);Proc. Natl. Acad. Sci. U.S.A., 77, 6114 (1980); J. Exp. Med., 155, 1133(1982); Proc. Natl. Acad. Sci. U.S.A., 81, 5767 (1984)).

KM-641 (FERM BP-3116) disclosed in EP-A-0 493 686 is an anti-GD₃monoclonal antibody belonging to the mouse IgG3 class, which reacts notonly with ganglioside GD₃ but also with ganglioside 3′,8′-LD1 and ispossessed of a broad range of antitumor spectrum. In addition, KM-641has stronger binding activities to antigens than anti-GD₃ monoclonalantibody R24 which has been disclosed in J. Exp. Med., 155, 1133 (1982)and it shows strong antitumor activities.

The mouse monoclonal antibody R24 to the ganglioside GD₃ was once usedfor the treatment of melanoma, but the administered mouse monoclonalantibody R24 did not fully exert its effect due to the formation ofanti-mouse immunoglobulin antibody in the patient's body (Eur. J. CancerClin. Oncol., 24, suppl 2, s 65 (1988)).

Consequently, the use of chimera antibody for anti-GD₃ monoclonalantibody would be advantageous in that anti-mouse immunoglobulinantibody does not form in the body, side effects are reduced oreliminated, its half life in blood is prolonged and its antitumoreffector effect increases, and thus therapeutic effects of the chimeraantibody which are superior to those of mouse monoclonal antibody can beobtained in the treatment of human cancers and the like.

Several processes for the production of humanized chimera antibodies areknown. Humanized chimera antibody, in which constant regions of theheavy chain (to be referred to as “H chain” hereinafter) and the lightchain (to be referred to as “L chain” hereinafter) of mouse monoclonalantibody are converted into human constant regions, is produced inanimal cells making use of recombinant DNA techniques. Examples of suchprocesses include a process in which humanized chimera antibody isproduced using chromosomal DNA as a gene which encodes mouse H chainvariable region (to be referred to as “V_(H)” hereinafter) and L chainvariable region (to be referred to as “V_(L)” hereinafter)(Morrison etal., Proc. Natl. Acad. Sci. U.S.A., 81, 6851 (1984); Neuberger et al.,Nature, 314, 268 (1985); Nishimura et al., Cancer Res., 47, 999 (1987);Dorai et al., J. Immunol., 139, 4232 (1987); Kameyama et al., FEBSletter, 244, 301 (1989)) and another process in which humanized chimeraantibody is produced using cDNA (Gillies et al., J. Immunol. Methods,125, 191 (1989); Liu et al., published International Application inJapan No. 2-501886). Cloning and base sequence determination ofhybridoma cell chromosomal DNA which encodes mouse V_(H) and V_(L)require much time and labor in comparison with those of cDNA thatencodes mouse V_(H) and V_(L). Consequently, the process in which cDNAis used for the production of humanized chimera antibody is moredesirable than the chromosomal DNA process.

Gillies et al. have succeeded in expressing humanized chimera antibodyin animal cells, making use of an expression vector for animal cellshaving inserted therein a humanized chimera H chain gene obtained bylinking mouse V_(H)-encoding cDNA with human C_(H)-encoding chromosomalDNA, and a humanized chimera L chain gene obtained by linking mouseV_(L)-encoding cDNA with human C_(L)-encoding chromosomal DNA (J.Immunol. Methods, 125, 191 (1989)). However, when an attempt was made toprepare chimera antibodies from several types of antibodies, a problemwas found that there were certain chimera antibodies whose L chainscould not be expressed without converting leader sequences. In addition,humanized chimera antibody can be produced more simply when cDNA whichencodes human C_(H) and C_(L) is used instead of the human C_(H)- andC_(L)-encoding chromosomal DNA.

In published International Application in Japan No. 2-501886, Liu et al.discloses a process for the expression of humanized chimera antibody inanimal cells, which comprises using an expression vector for animalcells having inserted therein a chimera H chain cDNA obtained by linkingmouse V_(H)-encoding cDNA with human C_(H)-encoding cDNA and a chimera Lchain cDNA obtained by linking mouse V_(L)-encoding cDNA with humanC_(L)-encoding cDNA. According to this process, however, it is necessaryto alter the J_(H) portion of the V_(H)-encoding cDNA and the J_(L)portion of the V_(L)-encoding cDNA by means of mutation, because thecDNA which encodes mouse V_(H) or V_(L) is linked with the human C_(H)-or C_(L)-encoding cDNA at the J region in the mouse variable region. Inaddition, with regard to the chimera L chain prepared using mouse Jk5,leucine which is one of the amino acids of the framework 4 is changed toisoleucine when made into humanized chimera antibody. Although aminoacid sequence of complementarity-determining region (to be referred toas “CDR” hereinafter) is especially important for antigen-antibodybinding, the amino acid sequence of the framework is also an importantfactor. For example, Riechmann et al. have prepared CDR graft antibodyby grafting a rat antibody CDR into a human antibody framework andreported that binding activity of the antibody was reduced by theframework conversion and the antibody activity increased when amino acidsequence of the framework was partially changed (Nature, 332, 323(1988)). Consequently, there is a possibility that the binding activityof humanized chimera antibody is undesirably reduced when the antibodyis produced by the mouse Jk5-aided process disclosed by Liu et al.

In view of the above, when any mouse antibody is converted intohumanized chimera antibody, it has been desired to simply and easilyproduce humanized chimera antibody in which amino acids of the mouseantibody variable region remain completely unchanged.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a process for theproduction of humanized chimera antibody by which the chimera antibodyis produced easily without changing any of the amino acids of its mouseantibody variable region. Another object of the present invention is toprovide a humanized chimera antibody to ganglioside GD₃ and a processfor the Production of such antibody.

SUMMARY OF THE INVENTION

The present invention relates to a process for producing humanizedchimera antibody which comprises the steps of:

-   (1) constructing a cassette vector by inserting a cDNA coding for    human antibody C_(H) into an expression vector for animal cell use    and establishing a cloning site in the upstream region C_(H) of said    cassette vector for inserting a cDNA which encodes nonhuman animal    V_(H);-   (2) digesting a cDNA coding for nonhuman animal antibody V_(H) with    restriction enzymes;-   (3) inserting said cDNA coding for nonhuman animal antibody V_(H)    into the cassette vector, using a synthetic DNA which comprises a    base sequence corresponding to the 5′-end side of said human    antibody C_(H) and a base sequence corresponding to the 3′-end side    of said nonhuman animal antibody V_(H) and is possessed of    restriction enzyme recognition sites on both of its ends, thereby    constructing a humanized chimera antibody H chain expression vector    in which said cDNA coding for human antibody C_(H) and said cDNA    coding for nonhuman animal antibody V_(H) are linked together    through said synthetic DNA;-   (4) constructing a cassette vector by inserting a cDNA coding for    human antibody C_(L) into an expression vector for animal cell use    and establishing a cloning site in the upstream region of the C_(L)    of said cassette vector for inserting a cDNA which encodes nonhuman    animal antibody V_(L);-   (5) digesting a cDNA coding for nonhuman animal antibody V_(L) with    restriction enzymes;-   (6) inserting said cDNA coding for nonhuman animal antibody V_(L)    into the cassette vector, using a synthetic DNA which comprises a    base sequence corresponding to the 5′-end side of said human    antibody C_(L) and a base sequence corresponding to the 3′-end side    of said nonhuman animal antibody V_(L) and is possessed of    restriction enzyme recognition sites on both of its ends, thereby    constructing a humanized chimera antibody L chain expression vector    in which said cDNA coding for human antibody C_(L) and said cDNA    coding for nonhuman animal antibody V_(L) are linked together    through said synthetic DNA;-   (7) introducing these expression vectors into host cells to obtain a    transformant; and-   (8) culturing said transformant in an appropriate culture medium,    thereby allowing the transformant to produce and accumulate a    humanized chimera antibody, and collecting said humanized chimera    antibody from the resulting culture broth.

The cassette vector to be used in the present invention is a vectorwhich is obtained by inserting a cDNA that encodes a constant region ofhuman antibody into an expression vector for animal cell use, in which acloning site is located in the upstream region of the constant regionfor inserting a cDNA that encodes a variable region of nonhuman animalantibody. An expression vector for humanized chimera antibody can beconstructed easily by inserting a variable region of nonhuman animalantibody into the cloning site of the cassette vector, using a syntheticDNA which comprises a base sequence corresponding to the 5′-end side ofa constant region of human antibody and a base sequence corresponding tothe 3′-end side of a variable region of nonhuman animal antibody and ispossessed of restriction enzyme recognition sites on its both ends.

The present invention also relates to a humanized chimera antibodyobtainable by the above-described process, a pharmaceutical compositioncomprising the humanized chimera antibody and a pharmaceuticallyacceptable carrier, and a method of treating cancer which comprisesadministering to a patient a pharmaceutically acceptable amount of saidhumanized chimera antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction enzyme cleavage map of a 9.3 kb XbaI fragmentof KM50 cell chromosomal DNA.

FIG. 2 shows a construction scheme for plasmid pKMB11.

FIG. 3 shows a construction scheme for plasmid pKMD6.

FIG. 4 shows a construction scheme for plasmid pEPKMA1.

FIG. 5 shows a construction scheme for plasmid pEPKMB1.

FIG. 6 shows a construction scheme for plasmid pAGE501.

FIG. 7 shows a construction scheme for plasmid pAGE109.

FIG. 8 shows a construction scheme for plasmid pAGE502.

FIG. 9 shows a construction scheme for plasmid pAGE503.

FIG. 10 shows a construction scheme for plasmid pSEd1.

FIG. 11 shows a construction scheme for plasmid pSE1D2.

FIG. 12 shows a construction scheme for plasmid pIG1SE1d2.

FIG. 13 shows a construction scheme for plasmid pIG1SE1d3.

FIG. 14 shows a construction scheme for plasmid pIG1SE1d4.

FIG. 15 shows a construction scheme for plasmid pPMOL2.

FIG. 16 shows a construction scheme for plasmid pPMOL3.

FIG. 17 shows a construction scheme for plasmid pchCKA7.

FIG. 18 shows a construction scheme for plasmid pchCKB1.

FIG. 18 shows a construction scheme for plasmid pckCKB1 (also SEQ IDNO:9).

FIG. 20 shows a construction scheme for plasmid pChiIgHB2.

FIG. 21 shows a construction scheme for plasmid pChiIgLA1.

FIG. 22 shows plasmids pKM641HA3 and pKM641LA2.

FIG. 23 shows plasmid pChi641HA1.

FIG. 24 shows a construction scheme for plasmid pKM641HE1.

FIG. 25 shows a construction scheme for plasmid pKM641HF1.

FIG. 26 shows a construction scheme for plasmid pChi641HA1.

FIG. 27 shows a construction scheme for plasmid pChi641HAM1.

FIG. 28 shows plasmid pChi641LG11.

FIG. 29 shows a construction scheme for plasmid pChi641LG11.

FIG. 30 shows a construction scheme for plasmid pChi641LGM11.

FIG. 31 shows a pattern of SDS-PAGE (4 to 15% gradient gel) of purifiedanti-GD₃ chimera antibody KM-871 (about 5 μg/lane) carried out underreductive condition (A) or non-reductive condition (B), where the lanesstarting from the left respectively indicate electrophoretic patterns ofmolecular weight markers, human IgG standard, mouse anti-GD₃ antibodyKM-641 and anti-GD₃ chimera antibody KM-871.

FIG. 32 is a graph showing reactivity of anti-GD₃ chimera antibodyKM-871 with ganglioside GD₃-positive G361 and SK-MEL-28 cells measuredby fluorescent antibody technique with the cell number on the ordinateand the fluorescence intensity on the abscissa. A dotted line showsreactivity in the absence of the antibody, while a solid line showsreactivity in the presence of KM-871.

FIG. 33 is a graph showing complement-dependent cytotoxicity (CDC) ofanti-GD₃ chimera antibody KM-871 and anti-GD₃ mouse antibody KM-641against ganglioside GD₃-positive G361 and SK-MEL-28 cells withcytotoxicity on the ordinate and an antibody concentration on theabscissa. A blackened bar shows CDC activity of KM-871, while a stripedbar shows that of KM-641.

FIG. 34 is a graph showing antibody-dependent cell-mediated cytotoxicity(ADCC) of KM-871 and KM-641 against ganglioside GD₃-positive cell G361with a ratio of effector cells to target cells on the ordinate and ADCCactivity on the abscissa. A blackened bar shows ADCC activity of KM-871,a dotted bar shows ADCC activity of KM-641 and a striped bar showscontrol (in the absence of the antibody). PMN means polymorphonuclearleukocyte.

FIG. 35 is a graph showing therapeutic effect of KM-871 on transplantedtumors with the tumor size on the ordinate and days aftertransplantation of tumors on the abscissa, in which closed circle showseffect of anti-Sialyl Le^(a) monoclonal antibody AMC-462, open squareshows that of KM-641 and open triangle shows that of KM-871.

DETAILED DESCRIPTION OF THE INVENTION

1. Construction of Cassette Vector

The cassette vector to be used in the present invention is constructedby inserting a cDNA which encodes a human antibody constant region intoan expression vector for animal cell use. Essential components in theexpression vector for animal cell use include promoter, enhancer, polyAsignal, splicing signal, drug resistance gene as a selection marker(e.g., ampicillin resistance gene, etc.) and the like. Any expressionvector for animal cell use may be used for this purpose, as long as itcan contain and express the cDNA molecule which encodes a human antibodyconstant region. For example, pAGE107 (Cytotechnology, 3, 133 (1990)) isuseful as such an expression vector. Examples of the promoter andenhancer for use in the expression vector for animal cell use include:SV40 early promoter and enhancer (J. Biochem., 101, 1307 (1987)); LTRpromoter and enhancer of Moloney mouse leukemia virus (Biochem. Biophys.Res. Comun., 149, 960 (1987)); and immunoglobulin H chain promoter(Cell, 41, 479 (1985)) and enhancer (Cell, 33, 717 (1983)). Theimmunoglobulin H chain promoter and enhancer can be prepared usingappropriate antibody-producing hybridoma cells, such as rat hybridomaKM50 cells which produce anti-human serum albumin antibody as disclosedin JP-A-60-258128 (the term “JP-A” as used herein means an “unexaminedpublished Japanese patent application”). The following describesprocesses for the preparation of the immunoglobulin H chain promoter andenhancer making use of KM50 cells.

Each chromosomal DNA is obtained from cultured KM50 cells, andP3X63Ag8U.1 (to be referred to as “P3U1” hereinafter) cells (ATCCCRL1597) which are to be fused with KM50 and rat kidney cells inaccordance with the procedure disclosed in Molecular Cloning (2nd. ed.,Cold Spring Harbor Laboratory Press, 1989, p9.14). Next, a DNA fragmentcontaining immunoglobulin promoter and enhancer and a gene of thevariable region of activated immunoglobulin H chain, in which DNArearrangement has been induced, is isolated from the chromosomal DNAextracted from KM50 cells, in accordance with the procedure disclosed inFEBS letter 244, 301 (1989). The immunoglobulin promoter and enhancerare cut out from the thus isolated DNA fragment and inserted into theaforementioned expression vector for animal cell use. Plasmid pIg1SE1d4is an illustrative example of the animal cell expression vector whichcontains the immunoglobulin H chain promoter and enhancer.

Next, a cloning site is established in the upstream region of a humanconstant region of a cassette vector, for inserting a cDNA which encodesa variable region of nonhuman animal antibody. Into the thus establishedcloning site is inserted a cDNA which encodes a variable region ofnonhuman animal antibody, using a synthetic DNA which comprises a basesequence corresponding to the 5′-end side of a constant region of humanantibody and a base sequence corresponding to the 3′-end side of avariable region of nonhuman animal antibody and is possessed ofrestriction enzyme recognition sites on both of its ends. In this way, ahumanized chimera antibody expression vector is constructed in which thecDNA coding for human antibody constant region and the cDNA coding forthe variable region of nonhuman animal antibody are linked togetherthrough the synthetic DNA. The synthetic DNA to be used may be preparedusing a DNA synthesizer, based on the base sequence which corresponds tothe 5′-end side of a constant region of human antibody and the basesequence that corresponds to the 3′-end side of a variable region ofnonhuman animal antibody. Illustrative examples of cloningsite-containing cassette vectors include a cassette vector pChiIgHB2which is used for the construction of an expression vector for theexpression of humanized chimera antibody H chain and a cassette vectorpChiIgLA2 which is used for the construction of an expression vector forthe expression of humanized chimera antibody L chain.

A cassette vector for use in the construction of an expression vectorfor the expression of humanized chimera antibody H chain is constructed,for example, by cutting out a human C_(H)-encoding cDNA-containingfragment, from an ApaI site in the vicinity of the 5′-end of the cDNA toits 3′-end, and inserting the fragment into an appropriate expressionvector for animal cell use such as plasmid pIg1SE1d4 or the like. Then,a cloning site is established in the thus constructed cassette vectorfor inserting a cDNA which encodes a V_(H) of nonhuman animal antibody.Into the thus established cloning site is then inserted a cDNA fragmentencoding a nonhuman animal antibody V_(H), which is obtained bydigesting a V_(H)-encoding cDNA with an appropriate restriction enzyme,using a synthetic DNA molecule which comprises a base sequencecorresponding to the 5′-end side (5′-end to ApaI site) of a humanantibody C_(H) and a base sequence corresponding to the 3′-end side of anonhuman animal antibody V_(H) and is possessed of restriction enzymerecognition sites on both of its ends. In this way, an expression vectorfor use in the expression of humanized chimera antibody H chain iseasily obtained without altering amino acid sequence of the expressedV_(H).

A cassette vector for constructing of an expression vector for theexpression of humanized chimera antibody L chain may be constructed forexample by introducing an EcoRV site into the vicinity of 5′-end side ofa human C_(L)-encoding cDNA by means of mutation, cutting out a fragmentfrom the resulting human cDNA From the EcoRV site to the 3′-end andinserting the fragment into an appropriate expression vector such asplasmid pIg1SE1d4 or the like. Then, a cloning site is established inthe thus constructed cassette vector for inserting a cDNA which encodesa nonhuman animal antibody V_(L). Into the thus established cloning siteis then inserted a cDNA fragment encoding a nonhuman animal antibodyV_(L), which is obtained by digesting a V_(L)-encoding cDNA with anappropriate restriction enzyme, using a synthetic DNA which comprises abase sequence corresponding to the 5′-end side (5′-end to EcoRV site) ofa human antibody C_(L) and a base sequence corresponding to the 3′-endside of a nonhuman animal antibody V_(L) and is possessed of restrictionenzyme recognition sites on both of its ends. In this way, an expressionvector for use in the expression of humanized chimera antibody L chainis easily obtained without altering amino acid sequence of the expressedV_(L).

Examples of the cDNAs which encode the human C_(H) and human C_(L)described above are disclosed, for instance, in Cell 22, 197 (1982).Such cDNAs can be prepared from human antibody-producing myeloma cells,humanized monoclonal antibody-producing hybridoma cells, humanizedchimera antibody-producing cells (SP2-PC chimera; FEBS Letters, 244, 301(1989)) and the like, in accordance with known procedures disclosed forinstance in Proc. Natl. Acad. Sci. U.S.A. 82, 7025 (1985) and ibid., 797025 (1985). That is, cDNA is synthesized using mRNA extracted from theabove-described cells, in accordance with the procedure disclosed inMolecular Cloning 2nd. ed.; 1989, p8.1. A library is prepared from thethus synthesized cDNA using a phage vector or a plasmid vector, inaccordance with the procedure disclosed in Molecular Cloning 2nd. ed.;1989, p8.1, 1.53. Next, a recombinant phage or a recombinant plasmidwhich contains human C_(H)-encoding cDNA or human C_(L)-encoding cDNA isobtained from the thus prepared library using a human antibody constantregion or a human antibody variable region as a probe, in accordancewith the procedure disclosed in Molecular Cloning 2nd. ed.; 1989, p8.1,1.53. Base sequences of the human C_(H)-encoding cDNA and the humanC_(L)-encoding cDNA are determined in accordance with the proceduredisclosed in Molecular Cloning, 2nd. ed.; 1989, p13.1. Introduction ofan appropriate restriction enzyme recognition site into the humanC_(L)-encoding cDNA, for example insertion of an EcoRV recognition siteinto a region in the vicinity of the 5′-end of the cDNA, may be effectedin accordance with the procedure disclosed in Molecular Cloning, 2nd.ed.; 1989, p15.1.

2. Production of Humanized Chimera Antibody

Firstly, cDNAs which encode V_(H) and V_(L) of nonhuman animal antibody,such as mouse anti-GD₃ monoclonal antibody, are prepared in thefollowing manner.

That is, cDNA is synthesized using mRNA extracted from appropriatehybridoma cells which produce mouse anti-GD₃ monoclonal antibody, suchas mouse anti-GD₃ monoclonal antibody KM-641 (FERM BP-3116). A libraryis prepared from the thus synthesized cDNA using a phage vector or aplasmid vector. Next, a recombinant phage or a recombinant plasmid whichcontains V_(H)-encoding cDNA or V_(L)-encoding cDNA is obtained from thethus prepared library using a constant region or a variable region ofnonhuman antibody, such as mouse antibody, as a probe. Base sequences ofthe V_(H)-encoding cDNA and the V_(L)-encoding cDNA are determined inaccordance with the aforementioned procedure.

A fragment of the V_(H)-encoding cDNA, ranging from the 5′-end to anappropriate restriction enzyme site near the 3′-end (to be referred toas “site A” hereinafter), is cut out and inserted into the cloning siteof the aforementioned cassette vector, using a synthetic DNA whichcomprises a base sequence corresponding to the 5′-end side of a humanantibody C_(H) and a base sequence corresponding to the 3′-end side(from 3′-end to site A) of a nonhuman animal antibody V_(H) and ispossessed of restriction enzyme recognition sites on both of its ends.In this way, an expression vector for use in the expression of humanizedchimera antibody H chain is constructed by linking the human antibodyC_(H)-encoding cDNA with the nonhuman antibody V_(H)-encoding cDNAthrough the synthetic DNA. In the same way, a fragment of theV_(L)-encoding cDNA, ranging from the 5′-end to an appropriaterestriction enzyme site near the 3′-end (to be referred to as “site B”hereinafter), is cut out and inserted into the cloning site of theaforementioned cassette vector, using a synthetic DNA molecule whichcomprises a base sequence corresponding to the 5′-end side of a humanantibody C_(L) and a base sequence corresponding to the 3′-end side(from 3′-end to site B) of a nonhuman animal antibody V_(L) and ispossessed of restriction enzyme recognition sites on both of its ends.In this way, an expression vector for use in the expression of humanizedchimera antibody L chain is constructed by linking the human antibodyC_(L)-encoding cDNA with the nonhuman antibody V_(L)-encoding cDNAthrough the synthetic DNA.

A transformant which is capable of producing humanized chimera antibodyis obtained by transforming appropriate host cells with the thusprepared expression vectors for use in the expression of the H chain andL chain of humanized chimera antibody.

Any type of cells may be used as host cells for use in the introductionof the humanized chimera antibody expression vectors, as long as thesecells are capable of expressing the humanized chimera antibody.Illustrative examples of such host cells include mouse SP2/0-Ag14 cells(ATCC CRL1581; to be referred to as “SP2/0 cells” hereinafter), mouseP3X63-Ag8.653 (ATCC CRL1580) and CHO cells which are deficient indihydrofolate reductase gene (to be referred to as “dhfr” hereinafter)(Urlaub et al., Proc. Natl. Acad. Sci. U.S.A., 77, 4216 (1980)).

Introduction of the expression vectors for use in the expression of theH chain and L chain of humanized chimera antibody into host cells may beeffected for example by the electroporation technique disclosed inJP-A-2-257891. A transformant capable of producing the humanized chimeraantibody may be selected using RPMI1640 medium supplemented with G418and fetal calf serum, in accordance with the procedure disclosed inJP-A-2-257891. A transformant, KM-871, which produces humanized chimeraantibody that reacts with ganglioside GD₃ is an illustrative example ofthe transformant capable of producing humanized chimera antibody. KM-871has been deposited on Aug. 13, 1991, with Fermentation ResearchInstitute, Agency of Industrial Science and Technology of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan under the Budapest Treaty, and hasbeen assigned the accession number FERM BP-3512.

For the cultivation of the thus-obtained transformants, any medium canbe used as long as the desired antibody can be produced and accumulatedin the medium. An example of such medium is RPMI1640 medium supplementedwith G418 and fetal calf serum. The transformants may be inoculated into200 μl to 100 ml of the above-mentioned medium to give a cellconcentration of 1×10⁵ to 1×10⁷ cells/ml and cultivated at 37° C. in a5% CO₂ incubator for 1 to 7 days. The desired chimera antibody isproduced and accumulated in the culture medium.

Activity of the humanized chimera antibody in the culture broth ismeasured by enzyme-linked immunosorbent assay (ELISA method; E. Harlowet al., Manual of Antibody Experiments, Cold Spring Harbor LaboratoryPress, 1988). Productivity of the humanized chimera antibody in thetransformant can be improved making use of a dhfr amplification systemin accordance with the procedure disclosed in JP-A-2-257891.

The humanized chimera antibody thus produced can be purified fromsupernatant fluid of the aforementioned cultured mixture making use of aprotein A column (E. Harlow et al., Manual of Antibody Experiments, ColdSpring Harbor Laboratory Press, 1988). Illustrative examples ofhumanized chimera antibodies obtained in this way include those whichreact with ganglioside GD₃, such as humanized chimera antibody EM-871and the like.

Reactivity of humanized chimera antibody is measured by ELISA method.The molecular weight of the H chain, the L chain or the entire moleculeof purified humanized chimera antibody is measured by means ofpolyacrylamide gel electrophoresis (SDS-PAGE), Western blotting method(E. Harlow et al., Manual of Ant-body Experiments, Cold Spring HarborLaboratory Press, 1988) or the like.

Binding activity, or avidity, of the humanized chimera antibody toganglioside GD₃ to a cultured cancer cell line is measured by means ofthe fluorescent antibody technique, the ELISA method or the like.Complement-dependent cytotoxicity (CDC activity) and antibody-dependentcell-mediated cytotoxicity (ADCC activity) of humanized chimera antibodyto a cultured cancer cell line are measured in accordance with theprocedures disclosed in Menekigaku Jikken Nyumon, (Manual ofImmunological Experiments) Matsuhashi et al., Gakkai Shuppan Center,Japan, 1981).

The humanized chimera antibodies according to the present invention canbe used alone as an anticancer agent. They may be formulated into ananticancer composition together with at least one pharmaceuticallyacceptable carrier. For instance, the humanized chimera antibodies aredissolved in physiological saline, an aqueous solution of glucose,lactose or mannitol and the like. The powder of the humanized chimeraantibodies for injection can be prepared by lyophilizing the humanizedchimera antibodies in accordance with the conventional method and mixingthe lyophilized products with sodium chloride. The anticancercomposition may further contain additives conventionally used well knownin the art of medical preparation, for example, pharmaceuticallyacceptable salts.

The humanized chimera antibodies according to the present invention canbe administered in the form of the above-described anticancercomposition to mammals including human in a dose of 0.2 to 20 mg/kg/day.The dose may vary depending on the age, condition, etc. of patients. Theadministration of the anticancer composition can be effected byintraveous injection once a day (single administration or consecutiveadministration) or intermittently one to three times a week or onceevery two to three weeks.

The antincancer composition is expected to be useful for treating cancersuch as melanoma, neuroblastoma and glioma.

The following examples and reference examples are provided to furtherillustrate the present invention. It is to be understood, however, thatthe examples are for purpose of illustration only and are not to beconstrued to limit the invention.

EXAMPLE 1

Construction of Cassette Vector

1. Isolation of Promoter and Enhancer Genes of KM50 Cell-derivedImmunoglobulin H Chain

(1) Preparation of Chromosomal DNA from KM50 Cells, P3U1 Cells and RatKidney

Chromosomal DNA was prepared in the following manner in accordance withthe procedure disclosed in Molecular Cloning, Maniatis et al., 1989,p9.16.

1.2×10⁸ KM50 cells, 2×10⁸ P3U1 cells (ATCC CRL1597) and 1.6 g of ratkidney (a kidney sample frozen at −80° C. was smashed thoroughly using amallet) were each suspended in 2 ml of a buffer solution (pH 7.5)containing 10 mM Tris-HCl, 150 mM sodium chloride and 10 mM sodiumethylenediaminetetraacetate (to be referred to as “EDTA” hereinafter).To this suspension were added 0.8 mg of Proteinase K (Sigma ChemicalCo.) and 10 mg of sodium lauryl sulfate (to be referred to as “SDS”hereinafter). After incubation at 37° C. for 10 hours, the resultingmixture was extracted with the same volume of phenol (once), chloroform(twice) and ether (once) in this order, and the extract was dialyzed for10 hours against a buffer solution (pH 7.5) containing 10 mM Tris-HCland 1 mM EDTA. A DNA solution was recovered from the dialysis tube andRibonuclease A (Sigma Chemical Co.) was added thereto to give a finalconcentration of 20 μg/ml. After incubating at 37° C. for 6 hours todecompose RNA completely, the resulting solution was mixed with 15 mg ofSDS and 1 mg of Proteinase K and incubated at 37° C. for 10 hours. Thethus treated solution was extracted with the same volume of phenol,chloroform and ether (twice for each) in this order, and the extract wasdialyzed for 10 hours against a buffer solution (pH 7.5) containing of10 mM Tris-HCl and 1 mM EDTA. The DNA solution was recovered from thedialysis tube and used as a chromosomal DNA sample. A DNA concentrationof each sample was determined by measuring the absorbance at 260 nm and,as a result, it was found that 1.6 mg, 1.5 mg and 1.9 mg of chromosomalDNA was obtained from 1.2×10⁸ KM50 cells, 2×10⁸ P3U1 cells and 1.6 g ofrat kidney, respectively.

(2) Identification of Activated Immunoglobulin H Chain Gene in KM50Cells by Southern Blotting

A 3 μg portion of each of the chromosomal DNA samples obtained in theabove step (1) from KM50 cells, P3U1 cells and rat kidney was dissolvedin 25 μl of a buffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mMmagnesium chloride and 100 mM sodium chloride. Each of the thus preparedsolution was mixed with 15 units of XbaI (Takara Shuzo Co., Ltd.; allrestriction enzymes used in the following experiments were purchasedfrom the same company) and incubated at 37° C. for 2 hours to cleave thechromosomal DNA at the XbaI site. The reaction mixture was subjected toagarose gel electrophoresis, resulting DNA fragments were transferred toa nitrocellulose filter in accordance with the method of Southern et al.(J. Mol. Biol., 98, 503, (1975)) and then subjected to hybridization inthe known method (Kameyama et al., FEBS Letters, 244, 301–306 (1989))using a mouse JH probe which is disclosed in the FEBS Letters article. Aband equivalent to about 9.3 kb was observed only in the DNA sample ofKM50 cells. In consequence, it was considered that the XbaI fragment ofimmunoglobulin DNA found in this band contained the activatedimmunoglobulin H chain gene derived from KM50 cells.

(3) Preparation of KM50 Cell Chromosomal DNA Library

A 60 μg portion of the KM50 cell chromosomal DNA obtained in the abovestep (2) was dissolved in 250 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 150 units of XbaI andincubated at 37° C. for 2 hours to cleave the chromosomal DNA at theXbaI site. The reaction mixture was subjected to agarose gelelectrophoresis and a 9.3 kb-equivalent fraction was recovered as about2 μg of 9.3 kb DNA sample of KM50 cells, making use of the DEAE papermethod (Maniatis et al., Molecular Cloning, 1989, p6.24). Separately, a3 μg portion of lambda-ZAP (Stratagene Cloning Systems) to be used as avector was dissolved in 200 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 50 units of XbaI and incubatedat 37° C. for 2 hours to cleave the DNA at the XbaI site. The resultingreaction mixture was extracted with phenol-chloroform and then treatedwith ethanol to precipitate and recover about 3 μg of DNA. The thusrecovered DNA sample was dissolved in a 100 μl of 100 mM Tris-HCl buffer(pH 7.5), and the resulting solution was mixed with 1 unit of alkalinephosphatase (Takara Shuzo Co., Ltd.) to effect dephosphorylation ofrestriction enzyme cleavage ends of the vector DNA. The resultingreaction mixture was extracted with phenol-chloroform and then treatedwith ethanol to precipitate and recover 2 μg of DNA. The thus recoveredDNA sample was dissolved in 10 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5) and 1 mM EDTA to serve as a vector DNA sample. Next,0.2 μg of the thus prepared vector DNA sample and 0.2 μg of the KM50cell-derived 9.3 kb DNA sample were dissolved in 5 μl of a buffersolution containing 66 mM Tris-HCl (pH 7.5), 6.6 mM magnesium chloride,10 mM dithiothreitol (to be referred to as “DTT” hereinafter) and 0.1 mMadenosine triphosphate (to be referred to as “ATP” hereinafter)(to bereferred to as “T4 ligase buffer” hereinafter). The resulting solutionwas mixed with 175 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) andincubated at 4° C. for 3 days. A 2 μl portion of the resulting reactionmixture was subjected to lambda phage packaging in the known method(Maniatis et al., Molecular Cloning, 1989, p2.95) using GigaPack Goldpurchased from Stratagene Cloning Systems. E. coli BB4 cells wereinfected with this phage to obtain 200,000 phage clones. 100,000 out ofthese phage clones were fixed on nitrocellulose filters in the knownmethod (Maniatis et al., Molecular Cloning, 1989, p2.112).

(4) Selection of Recombinant DNA Containing a Gene of the Activated(Anti-human Serum Albumin) Immunoglobulin H Chain Variable Region inKM50 Cells

Two clones showing strong reaction with the ³²P-labeled mouse JH probeat 65° C. were isolated from the 100,000 phage clones prepared in theabove step (3) in accordance with the procedure of Kameyama et al. (FEBSLetters, 44, 301–306, 1989). When the phage DNA was recovered in theconventional manner (Maniatis et al., Molecular Cloning, 1989,p2.118–2.169), it was found that the 9.3 kb XbaI fragment of the KM50cell-derived chromosomal DNA was incorporated into the phage DNA.

(5) Base Sequence of the Gene of the Activated (Anti-human SerumAlbumin) Immunoglobulin H Chain Variable Region in KM50 Cells

Restriction enzyme cleavage maps of the two clones obtained in the abovestep (4) was prepared by digesting them with various restriction enzymesand it was found that completely the same DNA fragment (9.3 kb) has beeninserted into these clones (FIG. 1). Next, base sequence of a part ofthe 9.3 kb DNA fragment, which was considered to contain the promoterand variable regions of the rat immunoglobulin H chain, was determinedin accordance with the Sanger method (Sanger et al., Proc. Natl. Acad.Sci. U.S.A., 74, 5463 (1977); M13 Cloning and Sequencing Handbook,Amersham). In SEQ ID NO: 1, a region containing octamer sequences suchas ATGCAAAT and TATA box sequences such as TTGAAAA and the like can beregarded as the immunoglobulin promoter region.

2. Construction of Heterologous Protein Expression Vector Using Promoterand Enhancer of the Activated (Anti-human Serum Albumin) ImmunoglobulinH Chain Variable Region in KM50 Cells

(1) Construction of pKMB11

A 1 μg portion of the 9.3 kb fragment of the immunoglobulin H chainvariable region gene obtained in 1-(5) was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride and 100 mM sodium chloride. The thus prepared solution wasmixed with 10 units of BglII and 10 units of HindIII and incubated at37° C. for 2 hours to cleave the DNA fragment at the BglII and HindIIIsites. The resulting reaction mixture was subjected to agarose gelelectrophoresis and 0.01 μg of a DNA fragment containing 0.8 kbimmunoglobulin promoter was recovered. Separately, a 1 μg portion of aplasmid pBR322-BglII (Kuwana et al., FEBS Letters, 219, 360 (1987)) wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 100 mM sodium chloride. The thusprepared solution was mixed with 10 units of BglII and 10 units ofHindIII and incubated at 37° C. for 2 hours to cleave the plasmid at theBglII and HindIII sites. The resulting reaction mixture was subjected toagarose gel electrophoresis, a DNA fragment of about 4.2 kb wasrecovered. A 0.1 μg portion of the thus obtained pBR322-BglII derivedDNA fragment of about 4.2 kb and 0.01 μg of the immunoglobulinpromoter-containing DNA fragment were dissolved in 20 μl of a T4 ligasebuffer, and the resulting solution was mixed with 175 units of T4 DNAligase (Takara Shuzo Co., Ltd.) and incubated at 4° C. for 24 hours.Using the resulting reaction mixture, transformation of E. coli HB101(J. Mol. Biol., 41, 459 (1969)) was carried out in accordance with themethod of Scott et al. (M. Shigesada, Saibo Kogaku, 2, 616 (1983)) toisolate a colony having ampicillin resistance (to be referred to as“ApR” hereinafter). Plasmid DNA was recovered from the colony to obtainpKMB11 as shown in FIG. 2.

(2) Construction of pKMD6

In order to establish an appropriate restriction enzyme recognition sitein downstream region of the immunoglobulin promoter, the plasmid pKMB11constructed in the above step (1) was digested wish nuclease BAL31 fromthe NcoI site. A 10 μg portion of the plasmid pKMB11 was dissolved in100 μl of a buffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mMmagnesium chloride and 50 mM potassium chloride. The thus preparedsolution was mixed with 30 units of NcoI and incubated at 37° C. for 2hours to cleave the plasmid at the NcoI site. The resulting reactionmixture was extracted with phenol and chloroform and treated withethanol. The thus precipitated DNA fragments were dissolved in 100 μl ofBAL31 buffer which contained 20 mM Tris-HCl (pH 8.0), 600 mM sodiumchloride, 12 mM calcium chloride, 12 mM magnesium chloride and 1 mMEDTA, and the resulting solution was mixed with 0.25 unit of BAL31(Bethesda Research Laboratories, Inc. (BRL)) and incubated at 37° C. for5 seconds. The reaction was stopped by extracting the reaction mixturewith phenol. After extraction with chloroform and precipitation withethanol, 1 μg of DNA was recovered. A 0.1 μg portion of the thusobtained DNA sample and 0.01 μg of a synthetic DNA linker SalI weredissolved in 20 μl of the T4 ligase buffer, and the resulting solutionwas mixed with 175 units of T4 DNA ligase and incubated at 4° C. for 24hours. Using the resulting reaction mixture, transformation of E. coliHB101 was carried out in accordance with the method of Scott et al. toisolate an ApR colony. Plasmid DNA was recovered from the colony toobtain pKMD6 as shown in FIG. 3. The base sequence of the BAL31-digestedportion of this plasmid was determined in accordance with the Sangermethod and it was found that bases up to the third base (the 303position base in the SEQ ID NO: 1) upstream from the initiation codonATG of the immunoglobulin gene.

(3) Construction of pEPKMA1, pEPKMB1 and pAGE501

Since original promoter and enhancer of the immunoglobulin gene areseparated from each other, it is necessary to construct a vector inwhich the promoter and enhancer are connected together so that it can beused as a vector for the expression of a heterologous protein. Thefollowing manipulation was carried out to construct such vectors.

A 1 μg portion of the 9.3 kb fragment of the immunoglobulin H chainvariable region gene obtained in 1-(5) was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride and 100 mM sodium chloride. The thus prepared solution wasmixed with 10 units of EcoRV and 10 units of XbaI and incubated at 37°C. for 2 hours to cleave the DNA fragment at the EcoRV and XbaI sites.The resulting reaction mixture was subjected to agarose gelelectrophoresis and 0.1 μg of a DNA fragment of about 1 kb containingthe immunoglobulin enhancer region was recovered. Separately, a 1 μgportion of the plasmid pKMD6 obtained in the above step (2) wasdissolved in 100 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 100 mM sodium chloride. The thusprepared solution was mixed with 10 units of BglII and incubated at 37°C. for 2 hours to cleave the plasmid at the BglII site. The resultingreaction mixture was extracted with phenol and chloroform andprecipitated with ethanol. The thus precipitated DNA fragments weredissolved in 40 μl of DNA polymerase I buffer containing 50 mM T=is HCl(pH 7.5), 10 mM magnesium chloride, 0.1 mM dATP (deoxyadenosinetriphosphate), 0.1 mM dCTP (deoxycytidine triphosphate), 0.1 mM dGTP(deoxyguanosine triphosphate) and 0.1 mM dTTP (deoxythymidinetriphosphate). The resulting solution was mixed with 6 units of E. coliDNA polymerase I Klenow fragment and incubated at 16° C. for 90 minutesto convert the cohesive 5′-end formed by the BglII digestion into bluntend. The reaction was stopped by extracting the reaction mixture withphenol. After extraction with chloroform and precipitation with ethanol,the resulting DNA fragments were dissolved in 30 ∥l of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mMsodium chloride. The thus prepared solution was mixed with 10 units ofHindIII and incubated at 37° C. for 2 hours to cleave the DNA fragmentat the HindIII site. The resulting reaction mixture was subjected toagarose gel electrophoresis, 0.1 μg of a DNA fragment of about 0.8 kbcontaining the immunoglobulin promoter region was recovered. Next, a 0.2μg portion of plasmid pUC18 (Messing, Methods in Enzymology, 101, 20(1983)) was dissolved in 30 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 10 units of HindIII and 10units of XbaI and incubated at 37° C. for 2 hours to cleave the plasmidat the HindIII and XbaI sites. The resulting reaction mixture wassubjected to agarose gel electrophoresis, 0.1 μg of a DNA fragment ofabout 2.7 kb was recovered. A 0.1 μg portion of the thus obtainedpKMD6-derived 0.8 kb DNA fragment, 0.02 μg of the DNA fragmentcontaining the immunoglobulin enhancer region and 0.1 μg of the pUC18fragment were dissolved in 20 μl of the T4 ligase buffer, and theresulting solution was mixed with 175 units of T4 DNA ligase andincubated at 4° C. for 24 hours. Using the resulting reaction mixture,transformation of E. coli HB101 was carried out to isolate an ApRcolony. Plasmid DNA was recovered from the colony to obtain pEPKMA1 asshown in FIG. 4.

Next, a 1 μg portion of the plasmid pEPKMA1 was dissolved in 100 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride and 100 mM sodium chloride. The thus prepared solution wasmixed with 10 units of XbaI and incubated at 37° C. for 2 hours tocleave the plasmid at the XbaI site. The resulting reaction mixture wasextracted with phenol and chloroform and precipitated with ethanol. Thethus precipitated DNA fragments were dissolved in 40 μl of theaforemetioned DNA polmerase I buffer solution, and the resultingsolution was mixed with 6 units of E. coli DNA polymerase I Klenowfragment and incubated at 16° C. for 90 minutes to convert the cohesive5′-end formed by the XbaI digestion into blunt end. The reaction wasstopped by extracting the reaction mixture with phenol. After extractionwith chloroform and precipitation with ethanol, DNA fragments wasrecovered. The thus contained DNA sample and 0.01 μg of a synthetic DNAXhoI linker (Takara Shuzo Co., Ltd.) were dissolved in 20 μl of the T4ligase buffer, and the resulting solution was mixed with 175 units of T4DNA ligase and incubated at 4° C. for 24 hours. Using the resultingreaction mixture, transformation of E. coli HB101 was carried out toisolate an ApR colony. Plasmid DNA was recovered from the colony toobtain pEPKMB1 as shown in FIG. 5.

Next, SV40 early gene promoter and enhancer regions (to be referred toas “P_(SE)” hereinafter) of an expression vector pAGE107 for use in theexpression of heterologous genes in animal cells (Miyaji et al.,Cytotechnology, 3, 133–140 (1990)) were converted into KM50-derivedimmunoglobulin H chain promoter and enhancer (to be referred to as“P_(IH)” hereinafter) of pEPKMB1 in the following manner.

A 1 μg portion of the plasmid pAGE107 was dissolved in 30 μl of a buffersolution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and150 mM sodium chloride. The thus prepared solution was mixed with 10units of SalI and 10 units of XhoI and incubated at 37° C. for 2 hoursto cleave the plasmid at the SalI and XhoI sites. The resulting reactionmixture was subjected to agarose gel electrophoresis and 0.5 μg of a DNAfragment of about 5.95 kb containing G418 resistance gene was recovered.Next, a 1 μg portion of the plasmid pEPKMB1 was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride and 150 mM sodium chloride. The thus prepared solution wasmixed with 10 units of SalI and 10 units of XhoI and incubated at 37° C.for 2 hours to cleave the plasmid at the SalI and XhoI sites. Theresulting reaction mixture was subjected to agarose gel electrophoresisand 0.1 μg of a DNA fragment of about 1.7 kb containing immunoglobulinpromoter and enhancer regions was recovered. A 0.1 μg portion of thethus obtained pAGE107-derived 5.95 kb DNA fragment and 0.02 μg of theDNA fragment containing immunoglobulin promoter and enhancer regionswere dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 175 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the resulting reaction mixture, transformation ofE. coli HB101 was carried out to isolate an ApR colony. Plasmid DNA wasrecovered from the colony to obtain pAGE501 as shown in FIG. 6.

(4) Construction of pAGE109

One of the two EcoRI cleavage sites in plasmid pAGE106 was deleted inthe following manner to construct pAGE109.

A 2 μg portion of the expression vector pAGE106 for use in theexpression of heterologous genes in animal cells (JP-A 3-22979 or EP-A-0405 285) was dissolved in 100 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mM sodium chloride.The thus prepared solution was mixed with 10 units of EcoRI and 10 unitsof SacI and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 1.5 μg ofa pAGE106 DNA fragment (4.3 kb) was recovered which contained the SV40early gene promoter and G418 resistance gene cleaved with EcoRI andSacI. The thus recovered DNA fragment was dissolved in 40 μl of the DNApolymerase I buffer solution, and the resulting solution was mixed with5 units of E. coli DNA polymerase I large fragment and incubated at 16°C. for 2 hours to convert the cohesive 3′-end formed by the SacIdigestion and the cohesive 5′-end formed by the EcoRI digestion intoblunt ends. The resulting reaction mixture was extracted with phenol andchloroform and then treated with ethanol. The thus precipitated samplewas dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 4 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain plasmidpAGE109 as shown in FIG. 7.

(5) Construction of pAGE502

Plasmid pAGE502 was constructed in the following manner in order toconvert the SV40 promoter and enhancer of pAGE107 into immunoglobulin Hchain promoter and enhancer.

A 2 μg portion of the plasmid pAGE107 disclosed in JP-A-3-22979 orEP-A-0 405 285 was dissolved in 100 μl of a buffer solution containing10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mM sodiumchloride. The thus prepared solution was mixed with 10 units of HindIII:and incubated at 37° C. for 4 hours. The resulting reaction mixture wassubjected to phenol-chloroform extraction and ethanol precipitation andthe thus recovered sample was dissolved in 40 μl of the DNA polymerase Ibuffer solution. The resulting solution was mixed with 5 units of E.coli DNA polymerase I Klenow fragment and incubated at 16° C. for 2hours to convert the cohesive 5′-end formed by the HindIII digestioninto blunt end. The resulting reaction mixture was extracted with phenoland chloroform and then treated with ethanol. The thus precipitatedsample was dissolved in 30 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 10 units of XhoI and incubatedat 37° C. for 4 hours. The resulting reaction mixture was subjected toagarose gel electrophoresis and about 1.5 μg of a pAGE107 DNA fragmentof about 5.95 kb was obtained which contained G418 resistance gene andApR gene cleaved with XhoI and HindIII.

Next, a 2 μg portion of the plasmid pAGE501 obtained in the above step(3) was dissolved in 100 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 175 mM sodium chloride.The thus prepared solution was mixed with 10 units of SalI and incubatedat 37° C. for 4 hours. After subjecting the resulting reaction mixtureto phenol-chloroform extraction and ethanol precipitation, the thusrecovered sample was dissolved in 40 μl of the DNA polymerase I buffersolution. The resulting solution was mixed with 5 units of E. coli DNApolymerase I Klenow fragment and incubated at 16° C. for 2 hours toconvert the cohesive 5′-end formed by the SalI digestion into blunt endThe resulting reaction mixture was extracted with phenol and chloroformand then treated with ethanol. The thus precipitated sample wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 100 mM sodium chloride. The thusprepared solution was mixed with 10 units of XhoI and incubated at 37°C. for 4 hours. The resulting reaction mixture was subjected to agarosegel electrophoresis and about 0.2 μg of a pAGE501 DNA fragment of about1.8 kb was obtained which contained KM50 immunoglobulin H chain promoterand enhancer genes cleaved with XhoI and SalI.

Next, 0.1 μg of the thus obtained pAGE107 HindIII-XhoI fragment (about5.95 kb) and 0.1 μg of the pAGE501 SalI-XhoI fragment (about 1.8 kb)were dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain plasmidpAGE502 as shown in FIG. 8.

(6) Construction of pAGE503

One of the two EcoRI cleavage sites in plasmid pAGE502 was deleted inthe following manner to construct pAGE503.

A 2 μg portion of the plasmid pAGE109 obtained in the above step (4) wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 50 mM sodium chloride. The thusprepared solution was mixed with 10 units of HindIII and 10 units ofClaI and incubated at 37° C. for 4 hours. The resulting reaction mixturewas subjected to agarose gel electrophoresis and about 0.2 μg of apAGE109 DNA fragment of about 1 kb was recovered which contained thepoly(A) signal gene of beta-globin and SV40 early genes cleaved withClaI and HindIII.

Next, a 2 μg portion of the plasmid pAGE502 obtained in the above step(5) was dissolved in 30 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mM sodium chloride.The thus prepared solution was mixed with 10 units of HindIII and 10units of ClaI and incubated at 37° C. for 4 hours. The resultingreaction mixture was subjected to agarose gel electrophoresis and thento the aforementioned DEAE paper method to recover about 1 μg of apAGE502 DNA fragment of about 6.1 kb which contained KM50 immunoglobulinH chain promoter and enhancer genes, ApR gene and G418 resistance genecleaved with HindIII and ClaI. Next, 0.1 μg of the thus obtained pAGE109HindIII-ClaI fragment (about 1 kb) and 0.1 μg of the pAGE502HindIII-ClaI fragment (about 6.1 kb) were dissolved in 20 μl of the T4ligase buffer, and the resulting solution was mixed with 350 units of T4DNA ligase and incubated at 4° C. for 24 hours. Using the thus obtainedrecombinant plasmid DNA, transformation of E. coli HB101 was carried outto obtain plasmid pAGE503 as shown in FIG. 9.

(7) Construction of pSE1d1

A dhfr gene was introduced into plasmid pAGE107 in the following mannerto construct plasmid pSE1d1.

A 2 μg portion of the plasmid pAGE107 disclosed in JP-A 3-22979 orEP-A-0 405 285 was dissolved in 100 μl of a buffer solution containing100 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mM sodiumchloride. The thus prepared solution was mixed with 10 units of EcoRIand incubated at 37° C. for 4 hours. After subjecting the resultingreaction mixture to phenol-chloroform extraction and ethanolprecipitation, the thus recovered sample was dissolved in 40 μl of theDNA polymerase I buffer solution. The resulting solution was mixed with5 units of E. coli DNA polymerase I Klenow fragment and incubated at 16°C. for 2 hours to convert the cohesive 5′-end formed by the EcoRIdigestion into blunt end. The resulting reaction mixture was extractedwith phenol and chloroform and then treated with ethanol. The thusprecipitated sample was dissolved in 30 μl of a buffer solution whichwas composed of 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50mM sodium chloride. The thus prepared solution was mixed with 10 unitsof HindIII and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 1.5 μg ofa pAGE107 DNA fragment of about 5.6 kb was recovered which containedG418 resistance gene and ApR gene cleaved with EcoRI and HindIII.

Next, a 2 μg portion of a plasmid pSV2-dhfr (Subramani et al., Mol.Cell. Biology, 1, 854 (1981)) was dissolved in 100 μl of a buffersolution containing 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and100 mM sodium chloride. The thus prepared solution was mixed with 10units of BglII and incubated at 37° C. for 4 hours. After subjecting theresulting reaction mixture to phenol-chloroform extraction and ethanolprecipitation, the thus recovered sample was dissolved in 40 μl of theDNA polymerase I buffer solution. The resulting solution was mixed with5 units of E. coli DNA polymerase I Klenow fragment and incubated at 16°C. for 2 hours to convert the cohesive 5′-end formed by the BglIIdigestion into blunt end. The resulting reaction mixture was extractedwith phenol and chloroform and then treated with ethanol. The thusprecipitated sample was dissolved in 30 μl of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mMsodium chloride. The thus prepared solution was mixed with 10 units ofHindIII and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis, about 0.2 μg of apSV2-dhfr DNA fragment of about 0.76 kb was recovered which containeddhfr gene cleaved with BglII and HindIII.

Next, 0.1 μg of the thus obtained pAGE107 HindIII-EcoRI fragment (about5.6 kb) and 0.1 μg of the pSV2-dhfr BglII-HindIII fragment (about 0.76kb) were dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain plasmid pSE1d1as shown in FIG. 10.

(8) Construction of pSE1d2

The HindIII cleavage site was removed from the plasmid pSE1d1 in thefollowing manner to construct plasmid pSE1d2.

A 2 μg portion of the plasmid pSE1d1 obtained in the above step (7) wasdissolved in 100 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 50 mM sodium chloride. The thusprepared solution was mixed with 10 units of HindIII and incubated at37° C. for 4 hours. After subjecting the resulting reaction mixture tophenol-chloroform extraction and ethanol precipitation, the thusrecovered sample was dissolved in 40 μl of the DNA polymerase I buffersolution. The resulting solution was mixed with 5 units of E. coli DNApolymerase I Klenow fragment and incubated at 16° C. for 2 hours toconvert the cohesive 5′-end formed by the HindIII digestion into bluntend. The resulting reaction mixture was extracted with phenol andchloroform and then treated with ethanol. The thus precipitated samplewas dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain plasmid pSE1d2as shown in FIG. 11.

(9) Construction of pIg1SE1d2

The dhfr gene was introduced into plasmid pAGE503 in the followingmanner to construct plasmid pIg1SE1d2.

A 2 μg portion of the plasmid pAGE503 obtained in the above step (6) wasdissolved in 100 μl of a buffer solution containing 100 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 50 mM sodium chloride. The thusprepared solution was mixed with 10 units of ClaI and incubated at 37°C. for 4 hours. After subjecting the resulting reaction mixture tophenol-chloroform extraction and ethanol precipitation, the thusrecovered sample was dissolved in 40 μl of the DNA polymerase I buffersolution. The resulting solution was mixed with 5 units of E. coli DNApolymerase I Klenow fragment and incubated at 16° C. for 2 hours toconvert the cohesive 5′-end formed by the ClaI digestion into blunt end.The resulting reaction mixture was extracted with phenol and chloroformand then treated with ethanol. The thus precipitated sample wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 50 mM sodium chloride. The thusprepared solution was mixed with 10 units of MluI and incubated at 37°C. for 4 hours. The resulting reaction mixture was subjected to agarosegel electrophoresis and about 1 μg of a pAGE503 DNA fragment of about5.4 kb was recovered which contained the KM50 immunoglobulin H chainpromoter and enhancer genes cleaved with ClaI and MluI.

Next, a 2 μg portion of the plasmid pSE1d2 obtained in the above step(8) was dissolved in 100 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 10 units of XhoI and incubatedat 37° C. for 4 hours. The resulting reaction mixture was subjected tophenol-chloroform extraction and ethanol precipitation and the thusrecovered sample was dissolved in 40 μl of the DNA polymerase I buffersolution. The resulting solution was mixed with 5 units of E. coli DNApolymerase I Klenow fragment and incubated at 16° C. for 2 hours toconvert the cohesive 5′-end formed by the XhoI digestion into blunt end.The resulting reaction mixture was extracted with phenol and chloroformand then treated with ethanol. The thus precipitated sample wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 6 mM magnesium chloride and 100 mM sodium chloride. The thusprepared solution was mixed with 10 units of MluI and incubated at 37°C. for 4 hours. The resulting reaction mixture was subjected to agarosegel electrophoresis and about 1 μg of a pSE1d2 DNA fragment of about 3.8kb was recovered which contained dhfr gene cleaved with XhoI and MluI.

Next, 1 μg of the thus obtained pAGE503 ClaI-MluI fragment (about 5.4kb) and 1 μg of the pSE1d2 XhoI-MluI fragment (about 3.8 kb) weredissolved in 20 μl of the T4 ligase buffer, and the resulting solutionwas mixed with 350 units of T4 DNA ligase and incubated at 4° C. for 24hours. Using the thus obtained recombinant plasmid DNA, transformationof E. coli HB101 was carried out to obtain plasmid pIg1SE1d2 as shown inFIG. 12.

(10) Construction of pIg1SE1d3

The ApaI cleavage site was removed from the plasmid pIg1SE1d2 in thefollowing manner to construct plasmid pIg1SE1d3.

A 2 μg portion of the plasmid pIg1SE1d2 obtained in the above step (9)was dissolved in 100 μl of a buffer solution containing 10 mM Tris-HCl(pH 7.5) and 6 mM magnesium chloride. The thus prepared solution wasmixed with 10 units of ApaI and incubated at 37° C. for 4 hours. Aftersubjecting the resulting reaction mixture to phenol-chloroformextraction and ethanol precipitation, the thus recovered sample wasdissolved in 40 μl of the DNA polymerase I buffer solution. Theresulting solution was mixed with 5 units of E. coli DNA polymerase IKlenow fragment and incubated at 16° C. for 2 hours to convert thecohesive 3′-end formed by the ApaI digestion into blunt end. Theresulting reaction mixture was extracted with phenol and chloroform andthen treated with ethanol. The thus precipitated sample was dissolved in20 μl of the T4 ligase buffer, and the resulting solution was mixed with350 units of T4 DNA ligase and incubated at 4° C. for 24 hours. Usingthe thus obtained recombinant plasmid DNA, transformation of E. coliHB101 was carried out to obtain plasmid pIg1SE1d3 as shown in FIG. 13.

(11) Construction of pIg1S1d4

In order to establish a cloning site between HindIII cleavage site andEcoRI cleavage site of the plasmid pIg1SE1d3, plasmid pIg1SE1d4 wasconstructed by inserting the synthetic DNA shown in SEQ ID NO:5 into theplasmid pIg1SE1d3 in the following manner.

A 2 μg portion of the plasmid pIg1SE1d3 obtained in the above step (10)was dissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl(pH 7.5), 6 mM magnesium chloride and 50 mM sodium chloride. The thusprepared solution was mixed with 10 units of HindIII and 10 units ofEcoRI and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 1 μg of apIg1SE1d3 DNA fragment of about 9.2 kb was recovered which contained theKM50 immunoglobulin H chain promoter and enhancer genes, ApR gene, G418resistance gene and dhfr gene cleaved with HindIII and EcoRI.

Next, 0.1 μg of the thus obtained pIg1SE1d3 HindIII-EcoRI fragment(about 9.2 kb) and 10 ng of the synthetic DNA (SEQ ID NO: 2) weredissolved in 20 μl of the T4 ligase buffer, and the resulting solutionwas mixed with 350 units of T4 DNA ligase and incubated at 4° C. for 24hours. Using the thus obtained recombinant plasmid DNA, transformationof E. coli HB101 was carried out to obtain plasmid pIg1SE1d4 as shown inFIG. 14.

3. Preparation of Moloney Mouse Leukemia Virus Long Terminal Repeat (tobe Referred to as “MoLTR” Hereinafter)

Since MoLTR is known to have promoter and enhancer activities (Kuwana etal., Biochem. Biophys. Res. Comun., 149, 960 (1987)), a plasmid pPMOL3containing MoLTR was prepared in the following manner in order to useMoLTR as cassette vector promoter and enhancer.

A 3 μg portion of the plasmid pPMOL1 disclosed in JP-A 1-63394 wasdissolved in 30 μl of a buffer solution containing 10 mM Tris-HCl (pH7.5), 7 mM magnesium chloride and 6 mM 2-mercaptoethanol. The thusprepared solution was mixed with 10 units of ClaI and incubated at 37°C. for 4 hours. After subjecting the resulting reaction mixture tophenol-chloroform extraction and ethanol precipitation, the thusrecovered sample was dissolved in 40 μl of the DNA polymerase I buffersolution. The resulting solution was mixed with 5 units of E. coli DNApolymerase I Klenow fragment and incubated at 16° C. for 2 hours toconvert the cohesive 5′-end formed by the ClaI digestion into blunt end.The reaction was stopped by phenol extraction, followed by chloroformextraction and ethanol precipitation to recover 2 μg of DNA fragments.The thus precipitated DNA sample and 0.01 μg of a synthetic DNA XhoIlinker (Takara Shuzo Co., Ltd.) were dissolved in 20 μl of the T4 ligasebuffer, and the resulting solution was mixed with 175 units of T4 DNAligase and incubated at 4° C. for 24 hours. Using the resulting reactionmixture, transformation of E. coli HB101 was carried out to obtainplasmid pPMOL2 as shown in FIG. 15. Next, a 3 μg portion of the thusobtained plasmid pPMOL2 was dissolved in 30 μl of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride, 10 mMsodium chloride and 6 mM 2-mercaptoethanol. The thus prepared solutionwas mixed with 10 units of SmaI and incubated at 37° C. for 4 hours. Theresulting reaction mixture was subjected to phenol-chloroform extractionand ethanol precipitation and 2 μg of DNA fragments were recovered. Thethus recovered DNA sample and 0.01 μg of a synthetic DNA EcoRI linker(Takara Shuzo Co., Ltd.) were dissolved in 20 μl of the T4 ligasebuffer, and the resulting solution was mixed with 175 units of T4 DNAligase and incubated at 4° C. for 24 hours. Using the resulting reactionmixture, transformation of E. coli HB101 was carried out to obtainplasmid pPMOL3 as shown in FIG. 16.

4. Cloning of H Chain Constant Region (Cg1) cDNA and L Chain ConstantRegion (Ck) cDNA of Human Immunoglobulin IgG1

(1) Preparation of mRNA from Chimera Antibody-producing SP2-PC Chimera-1Cells

Using a mRNA extraction kit, Fast Track (No. K1593-02, available fromInvitrogen), 6.2 μg of mRNA was obtained from 1×10⁸ cells of chimeraantibody-producing SP2-PC Chimera-1 which has anti-phosphorylcholineactivity and is disclosed in FEBS Letters (244, 301–306 (1989)).

(2) Preparation of SP2-PC Chimera-1 cDNA Library and Cloning of HumanImmunoglobulin H Chain Constant Region (Cg1) cDNA and L Chain ConstantRegion (Ck) cDNA

A 2 μg portion of the mRNA obtained in the above step (1) was subjectedto EcoRI adaptor addition using cDNA Synthesis Kit (No. 27-9260-01,available from Pharmacia) followed by kination. The resulting cDNAsolution was subjected to phenol-chloroform extraction and ethanolprecipitation to recover 4 μg of cDNA. The thus recovered cDNA wasdissolved in 20 μl of sterile water, and the resulting solution wassubjected to agarose gel electrophoresis to recover about 0.3 μg of aDNA fragment of about 1.8 kb and about 0.3 μg of a DNA fragment of about1.0 kb.

Next, a 5 μg portion of the vector pUC18 was dissolved in 100 μl of abuffer solution containing 100 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride and 100 mM sodium chloride. The thus prepared solution wasmixed with 50 units of EcoRI and incubated at 37° C. for 4 hours tocleave the pUC18 DNA at its EcoRI cleavage site. The resulting reactionmixture was subjected to phenol-chloroform extraction and ethanolprecipitation to recover about 3 μg of a pUC18 DNA fragment cleaved withEcoRI.

Next, 0.1 μg of the thus obtained pUC18 EcoRI fragment (about 2.7 kb)and the 1.8 kb and 1.0 kb cDNA fragments (0.1 μg for each) prepared fromthe SP2-PC Chimera-1 cells were dissolved in 20 μl of the T4 ligasebuffer, and the resulting solution was mixed with 350 units of T4 DNAligase and incubated at 4° C. for 24 hours.

Using the thus obtained recombinant plasmid DNA, transformation of E.coli LE392 was carried out. About 3,000 colonies thus obtained werefixed on nitrocellulose filters. Two ³²P-labeled probes were preparedfrom human immunoglobulin constant region chromosomal genes (Cg1 as anIgG1 H chain constant region and Ck as an IgG1 L chain constant region)which have been isolated by Kameyama et al. (FEBS Letters, 244, 301(1989)). From colonies which showed strong reactions at 65° C. withthese probes, one showing strong reaction with Cg1 (pPCVHhCGI1) and theother showing strong reaction with Ck (pPCVLhCK1) were obtained.

(3) Introduction of EcoRV Site into Human Ig k Chain Constant Region

An EcoRV site was introduced into 5′-end side of the human Ig k chainconstant region by means of site-specific mutagenesis using a kitpurchased from Promega (Catalogue No. Q6210). A 2 μg portion of theplasmid pPCVLhCK1 was dissolved in 30 μl of a buffer solution containing10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mM sodiumchloride. The thus prepared solution was mixed with 10 units of EcoRIand 10 units of KpnI and incubated at 37° C. for 4 hours. The resultingreaction mixture was subjected to agarose gel electrophoresis and about0.2 μg of a pPCVLhCK1 DNA fragment of about 0.8 kb was recovered whichcontained the human immunoglobulin L chain constant region cleaved withEcoRI and KpnI.

Next, a 2 μg portion of pSELECT1 (a kit available from Promega,Catalogue No. Q6210) was dissolved in 30 μl of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 50 mMsodium chloride. The thus prepared solution was mixed with 10 units ofEcoRI and 10 units of KpnI and incubated at 37° C. for 4 hours. Theresulting reaction mixture was subjected to agarose gel electrophoresisand about 1 μg of a pSELECT1 DNA fragment of about 5.7 kb cleaved withEcoRI and KpnI was recovered.

Next, 0.1 μg of the pPCVLhCK1 EcoRI-KpnI fragment (about 0.8 kb) and 0.1μg of the pSELECT1 EcoRI-KpnI fragment (about 5.7 kb) obtained abovewere dissolved in 20 μl of the T4 ligase buffer, and the resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli JM109 was carried out to obtain plasmidpchCKA7 as shown in FIG. 17.

Next, using the plasmid pchCKA7 thus obtained and the synthetic DNA ofSEQ ID NO:6 as mutagenesis primer, the ACC sequence of the humanimmunoglobulin L chain constant region (12 to 14 position bases from theN-terminal) was converted into GAT in order to construct a plasmidpchCKB1 (FIG. 18) in which an EcoRV site was introduced into theconverted site.

Next, the EcoRV site of the plasmid pchCKB1 was converted into HindIIIcleavage site in the following manner. A 2 μg portion of the plasmidpchCKB1 obtained above was dissolved in 10 μl of a buffer solutioncontaining 100 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mMsodium chloride. The thus prepared solution was mixed with 10 units ofEcoRI and incubated at 37° C. for 4 hours. After subjecting theresulting reaction mixture to phenol-chloroform extraction and ethanolprecipitation, the thus recovered sample was dissolved in 40 μl of theDNA polymerase I buffer solution. The resulting solution was mixed with5 units of E. coli DNA polymerase I Klenow fragment and incubated at 37°C. for 30 minutes to convert the cohesive 5′-end formed by the EcoRIdigestion into blunt end. The resulting reaction mixture was extractedwith phenol and chloroform and then treated with ethanol. The thusprecipitated sample was dissolved in 20 μl of the T4 ligase buffercontaining 0.1 μg of HindIII linker (Takara Shuzo Co., Ltd.), and theresulting solution was mixed with 350 units of T4 DNA ligase andincubated at 4° C. for 24 hours. Using the thus obtained recombinantplasmid DNA, transformation of E. coli HB101 was carried out to obtainplasmid pchCKC1 as shown in FIG. 19.

5. Construction of Cassette Vector

(1) Construction of a Cassette Vector for use in the Construction ofHumanized Chimera Antibody H Chain Expression Vector

A 2 μg portion of the plasmid pIg1SE1d4 obtained in the aforementionedstep 2-(11) was dissolved in 30 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 10 units of EcoRV and 10 unitsof ApaI and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 1.5 μg ofa pIg1SE1d4 DNA fragment of about 9.2 kb cleaved with EcoRV and ApaI wasrecovered.

Next, a 2 μg portion of the plasmid pPCVHhCGI1 obtained in theaforementioned step 4-(2) was dissolved in 30 μl of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5) and 6 mM magnesium chloride. The thusprepared solution was mixed with 10 units of ApaI and 10 units of SmaIand incubated at 37° C. for 1 hour. The resulting reaction mixture wassubjeced to agarose gel electrophoresis and about 0.2 μg of a pPCVHhCGI1DNA fragment of about 1 kb was recovered which contained the humanimmunoglobulin H chain constant region gene cleaved with ApaI and SmaI.

Next, 0.1 μg of the pIg1SE1d4 EcoRV-ApaI fragment (about 9.2 kb) and 0.1μg of the pPCVHhCGI1 ApaI-SmaI fragment (about 1 kb) prepared above weredissolved in 20 μl of the T4 ligase buffer. The resulting solution wasmixed with 350 units of T4 DNA ligase and incubated at 4° C. for 24hours. Using the thus obtained recombinant plasmid DNA, transformationof E. coli HB101 was carried out to obtain a plasmid pChiIgHB2 (FIG. 20)as a cassette vector for use in the construction of a humanized chimeraantibody H chain expression vector.

(2) Construction of a Cassette Vector for use in the Construction ofHumanized Chimera Antibody L Chain Expression Vector

A 2 μg portion of the plasmid pIg1SE1d4 obtained in the aforementionedstep 2-(11) was dissolved in 30 μl of a buffer solution containing 10 mMTris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mM sodium chloride.The thus prepared solution was mixed with 10 units of EcoRV and 10 unitsof HindIII and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 1.5 μg ofa pIg1SE1d4 DNA fragment of about 9.2 kb cleaved with EcoRV and HindIIIwas recovered.

Next, a 2 μg portion of the plasmid pchCKC1 obtained in theaforementioned step 4-(3) was dissolved in 30 μl of a buffer solutioncontaining 10 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride and 100 mMsodium chloride. The thus prepared solution was mixed with 10 units ofEcoRV and 10 units of HindIII and incubated at 37° C. for 1 hour. Theresulting reaction mixture was subjected to agarose gel electrophoresisand about 0.2 μg of a pPCVLhCK1 DNA fragment of about 0.6 kb wasrecovered which contained the human immunoglobulin L chain constantregion gene cleaved with EcoRV and HindIII.

Next, 0.1 μg of the pIg1SE1d4 EcoRV-HindIII fragment (about 9.2 kb) and0.1 μg of the pchCKC1 EcoRV-HindIII fragment (about 0.6 kb) preparedabove were dissolved in 20 μl of the T4 ligase buffer. The resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain a plasmidpChiIgLA1 (FIG. 21) as a cassette vector for use in the construction ofa humanized chimera antibody L chain expression vector.

EXAMPLE 2

Anti-GD₃ Chimera Antibody

1. Preparation of mRNA from a Hybridoma Cell Line Capable of ProducingMouse Anti-GD₃ Monoclonal Antibody KM-641

Using a mRNA extraction kit, Fast Track (No. K1593-02, available fromInvitrogen), 34 μg of mRNA was prepared from 1×10⁸ cells of a hybridomacell line which is capable of producing mouse anti-GD₃ monoclonalantibody KM-641 prepared by the method described later in ReferenceExample shown below.

2. Preparation of cDNA Libraries of KM-641 H Chain and L Chain Genes

Using ZAP-cDNA Synthesis Kit (No. sc200400), a cDNA synthesis kitavailable from Stratagene Cloning Systems, cDNA having EcoRI adaptor onits 5′-end and XhoI adaptor on its 3′-end was prepared from 3 μg of themRNA obtained in the above procedure 1. About 6 μg of the cDNA wasdissolved in 10 μl of sterile water and subjected to agarose gelelectrophoresis to recover 0.1 μg of an H chain-corresponding cDNAfragment of about 1.8 kb and 0.1 μg of an L chain-corresponding cDNAfragment of about 1.0 kb. Next, 0.1 μg of the 1.8 kb cDNA fragment, 0.1μg of the 1.0 kb cDNA fragment and 1 μg of Uni-ZAP XR (available fromStratagene Cloning Systems; a preparation obtained by digesting LambdaZAPII vector with EcoRI and XhoI, followed by treatment with calfintestine alkaline phosphatase) to be used as a vector were dissolved in11.5 μl of the T4 ligase buffer, and the resulting solution was mixedwith 175 units of T4 DNA ligase and incubated at 12° C. for 10 hours andthen at room temperature for 2 hours. A 4 μl portion of the resultingreaction mixture was subjected to lambda phage packaging using Giga PackGold (Stratagene Cloning Systems) in accordance with the conventionalmethod (Maniatis et al., Molecular Cloning, 1989, p2.95). An E. colistrain PLK-F was infected with the thus packaged product in accordancewith the conventional method (Maniatis et al., Molecular Cloning, 1989,p2.95–107) to obtain an H chain cDNA library and an L chain cDNAlibrary, each containing about 10,000 phage clones. Next, these phageparticles were fixed on nitrocellulose filters in accordance with theconventional method (Maniatis et al., Molecular Cloning, 1989, p2.112).

3. Cloning of Monoclonal Antibody KM-641 H Chain and L Chain cDNA

Two ³²P-labeled probes were prepared from an EcoRI fragment of about 6.8kb containing a mouse immunoglobulin constant region chromosomal geneCg1 (Roeder et al., Proc. Natl. Acad. Sci. U.S.A., 78, 474 (1981)) and amouse Ck gene-containing HindIII-BamHI fragment of about 3 kb (Sakano etal., Nature, 280, 288 (1979)). A phage clone which showed strongreaction at 65° C. with one of these two probes were obtained from eachof the H chain cDNA library and the L chain cDNA library prepared in theabove procedure 2 in accordance with the conventional method (Maniatiset al., Molecular Cloning, 1989, p2.108). Next, using ZAP-cDNA SynthesisKit (No. sc200400), a cDNA synthesis kit of Stratagene Cloning Systemseach of the thus obtained phage clones was introduced into plasmidpBluescript to isolate a recombinant plasmid pKM641HA3 containing theKM-641 H chain cDNA and a recombinant plasmid pKM641LA2 containing theKM-641 L chain cDNA. When each of the plasmids pKM641HA3 and pKM641LA2was digested with EcoRI and XhoI, it was found that cDNA of about 1.6 kbhad been inserted into the former plasmid, and cDNA of about 0.9 kb intothe latter (FIG. 22).

4. Immunoglobulin Variable Region Base Sequences of KM-641 H Chain cDNA(pKM641HA3) and KM-641 L Chain cDNA (pKM641LA2)

Immunoglobulin variable region base sequences of the plasmids pKM641HA4and pKM641LA2 obtained in the above procedure 3 were determined by thedideoxy method (Maniatis et al., Molecular Cloning, 1989, p13.42) usingSequenase Version 2.0 DNA Sequencing Kit (United States BiochemicalCorporation). The results are shown in SEQ ID NO:7 and SEQ ID NO:9. Theplasmid pKM641LA2 was a complete DNA containing a leader sequence andhaving a methionine-corresponding sequence which was assumed to be theinitiation codon ATG located close to the 5′-end. The plasmid pKM641HA3,on the other hand, did not have such a methionine-correspondinginitiation codon-like sequence on its 5′-end side, and its leadersequence was partially deficient.

5. Construction of KM-641 Chimera H Chain Expression Vector

H chain variable region gene obtained by cleaving the plasmid pKM641HA3variable region at the 5′-end AluI site and 3′-end StyI site was ligatedwith the cassette vector for use in the construction of the humanizedchimera antibody H chain obtained in Example 1 using the synthetic DNAsequences shown in SEQ ID NO:11 and SEQ ID NO:13, thereby constructing ahumanized chimera antibody H chain expression vector pchi641HA1 (FIG.23).

Firstly, the DNA shown in SEQ ID NO:13 (see FIG. 23) was synthesizedusing a DNA synthesizer. This synthetic DNA comprises a base sequencederived from plasmid pKM641HA3 ranging from the 3′-end of itsimmunoglobulin H chain variable region to a StyI cleavage site in thevicinity of the 3′-end and a base sequence derived from plasmid pAGE28ranging from the 5′-end of its immunoglobulin H chain constant region toan ApaI cleavage site in the vicinity of the 5′-end. Thus, the syntheticDNA has a StyI cleavage site and an ApaI cleavage site on both of itsend. Next, the thus synthesized DNA was introduced into the plasmidpKM641HA3 in the following manner.

A 3 μg portion of the plasmid pKM641HA3 was dissolved in 30 μl of abuffer solution containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT. The thus prepared solutionwas mixed with 10 units of EcoRI and 10 units of StyI and incubated at37° C. for 4 hours. The resulting reaction mixture was subjected toagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about0.41 kb was recovered. Next, a 3 μg portion of pAGE28 (Mizukami et al.,J. Biochem., 101, 1307–1310 (1987)) was dissolved in 30 μl of a buffersolution containing 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride and6 mM 2-mercaptoethanol. The thus prepared solution was mixed with 10units of EcoRI and 10 units of ApaI and incubated at 37° C. for 4 hours.The resulting reaction mixture was subjected to agarose gelelectrophoresis and about 2 μg of a DNA fragment of about 2.45 kb wasrecovered. Next, 0.1 μg of the pKM641HA3 EcoRI-StyI fragment (about 0.41kb) and 0.1 μg of the pAGE28 EcoRI-ApaI fragment (about 2.45 kb)prepared above and 0.3 μg of the synthetic DNA of SEQ ID NO: 13 weredissolved in 20 μl of the T4 ligase buffer solution. The resultingsolution was mixed with 350 units of T4 DNA ligase and incubated at 4°C. for 24 hours. Using the thus obtained recombinant plasmid DNA,transformation of E. coli HB101 was carried out to obtain a plasmidpKM641HE1 as shown in FIG. 24.

Since the thus constructed plasmid pKM641HE1 lacks a leader sequence,the following attempt was made to supplement the plasmid with the leadersequence using the synthetic DNA of SEQ ID NO:11.

A 3 μg portion of the plasmid pKM641HE1 was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 7 mM magnesiumchloride and 6 mM 2-mercaptoethanol. The thus prepared solution wasmixed with 10 units of EcoRI and 10 units of ApaI and incubated at 37°C. for 4 hours. The resulting reaction mixture was subjected to agarosegel electrophoresis and about 0.4 μg of a DNA fragment of about 0.42 kbwas recovered. Next, a 0.4 μg portion of the thus prepared pKM641HE1EcoRI-ApaI fragment (about 0.42 kb) was dissolved in 30 μl of a buffersolution containing 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride, 50mM sodium chloride and 6 mM 2-mercaptoethanol. The thus preparedsolution was mixed with 10 units of AluI and incubated at 37° C. for 4hours. The resulting reaction mixture was subjected to phenol-chloroformextraction and ethanol precipitation and about 0.3 μg of a DNA fragmentof about 0.4 kb was recovered.

Next, 0.1 μg of the pKM641HE1 AluI-ApaI fragment (about 0.4 kb) and 0.1μg of the pAGE28 EcoRI-ApaI fragment (about 2.45 kb) prepared above and0.3 μg of the synthetic DNA of SEQ ID NO:11 were dissolved in 20 μl ofthe T4 ligase buffer solution. The resulting solution was mixed with 350units of T4 DNA ligase and incubated at 4° C. for 24 hours. Using thethus obtained recombinant plasmid DNA, transformation of E. coli HB101was carried out to obtain a plasmid pKM641HF1 as shown in FIG. 25.

Next, immunoglobulin H chain variable region of the thus obtainedplasmid pKM641HF1 was introduced into the aforementioned cassette vectorpChiIgHB2 in the following manner.

A 3 μg portion of the plasmid pKM641LA2 was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 7 mM magnesiumchloride, 50 mM sodium chloride and 6 mM 2-mercaptoethanol. The thusprepared solution was mixed with 10 units of EcoRI and 10 units ofHindIII and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 0.3 μg ofa DNA fragment of about 0.35 kb was recovered. Next, a 3 μg portion ofpChiIgLA1 was dissolved in 30 μl of a buffer solution containing 50 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride and 1mM DTT. The thus prepared solution was mixed with 10 units of EcoRI and10 units of EcoRV and incubated at 37° C. for 4 hours. The resultingreaction mixture was subjected to phenol-chloroform extraction andethanol precipitation and about 3 μg of DNA was recovered and dissolvedin 10 μl of the TE solution (a buffer solution containing 10 mM Tris-HCland 1 mM EDTA (pH 7.7). Next, 0.1 μg of the pKM641LA2 EcoRI-HindIIIfragment (about 0.35 kb) and 0.1 μg of the pChiIgLA1 EcoRI-EcoRVfragment (about 9.7 kb) prepared above and 0.3 μg of the synthetic DNAof SEQ ID NO:15 were dissolved in 20 μl of the T4 ligase buffersolution. The resulting solution was mixed with 350 units of T4 DNAligase and incubated at 4° C. for 24 hours. Using the thus obtainedrecombinant plasmid DNA, transformation of E. coli HB101 was carried outto obtain a plasmid pChi641LG11 as shown in FIG. 29.

Next, KM50-derived immunoglobulin H chain promoter and enhancer regionsof the thus obtained plasmid pChi641HA1 were converted into MoLTR in thefollowing manner.

A 3 μg portion of the plasmid pChi641HA1 was dissolved in 30 μl of abuffer solution containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT. The thus prepared solutionwas mixed with 10 units of EcoRI and 10 units of XhoI and incubated at37° C. for 4 hours. The resulting reaction mixture was subjected toagarose gel electrophoresis and about 0.2 μg of a DNA fragment of about8.8 kb was recovered. Next, a 3 μg portion of the pPMOL3 prepared inprocedure 2 of Example 1 was dissolved in 30 μl of a buffer solutioncontaining 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mMsodium chloride and 1 mM DTT. The thus prepared solution was mixed with10 units of EcoRI and 10 units of XhoI and incubated at 37° C. for 4hours. The resulting reaction mixture was subjected to agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.63 kbcontaining MoLTR was recovered. Next, 0.1 μg of the pChi641HA1EcoRI-XhoI fragment and 0.1 μg of the pPMOL3 EcoRI-XhoI fragmentprepared above were dissolved in 20 μl of the T4 ligase buffer solution.The resulting solution was mixed with 175 units of T4 DNA ligase andincubated at 4° C. for 24 hours. Using the resulting reaction mixture,transformation of E. coli HB101 was carried out to obtain a plasmidpChi641HAM1 (FIG. 27) as a KM-641 chimera H chain expression vector.

6. Construction of KM-641 Chimera L Chain Expression Vector

L chain variable region gene obtained by cleaving the plasmid pKM641LA2variable region gene at its 5′-end EcoRI site and 3′-end HindIII sitewas ligated with the cassette vector for the expression of chimera Lchain, using the synthetic DNA shown in SEQ ID NO: 15, therebyconstructing an L chain expression vector pchi641LG11 (FIG. 28).

Firstly, the DNA of SEQ ID NO: 8 (see FIG. 29) was synthesized using aDNA synthesizer. This synthetic DNA comprises a base sequencecorresponding to a region of the plasmid pKM641LA2 ranging from the3′-end of the immunoglobulin L chain variable region to a HindIIIcleavage site in the vicinity of the 3′-end and a base sequencecorresponding to a region of the plasmid pChiIgLA1 ranging from the5′-end to an EcoRV cleavage site in the vicinity of the 5′-end. Thus, ithas a HindIII cleavage site and an EcoRV cleavage site on both ends.Next, the thus synthesized DNA was introduced into the plasmid pKM641LA2in the following manner.

A 3 μg portion of the plasmid pKM641LA2 was dissolved in 30 μl of abuffer solution containing 10 mM Tris-HCl (pH 7.5), 7 mM magnesiumchloride, 50 mM sodium chloride and 6 mM 2-mercaptoethanol. The thusprepared solution was mixed with 10 units of EcoRI and 10 units ofHindIII and incubated at 37° C. for 4 hours. The resulting reactionmixture was subjected to agarose gel electrophoresis and about 0.3 μg ofa DNA fragment of about 0.35 kb was recovered. Next, a 3 μg portion ofpChiIgLA1 was dissolved in 30 μl of a buffer solution containing 50 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride and 1mM DTT. The thus prepared solution was mixed with 10 units of EcoRI and10 units of EcoRV and incubated at 37° C. for 4 hours. The resultingreaction mixture was subjected to phenol-chloroform extraction andethanol precipitation and about 3 μg of DNA was recovered and dissolvedin 10 μl of the TE solution (a buffer solution containing 10 mM Tris-HCland 1 mM EDTA (pH 7.5)). Next, 0.1 μg of the pKM641LA2 EcoRI-HindIIIfragment (about 0.35 kb) and 0.1 μg of the pChiIgLA1 EcoRI-EcoRVfragment (about 9.7 kb) prepared above and 0.3 μg of the synthetic DNAof SEQ ID NO:8 were dissolved in 20 μl of the T4 ligase buffer solution.The resulting solution was mixed with 350 units of T4 DNA ligase andincubated at 4° C. for 24 hours. Using the thus obtained recombinantplasmid DNA, transformation of E. coli HB101 was carried out to obtain aplasmid pChi641LG11 as shown in FIG. 29.

Next, KM50-derived immunoglobulin H chain promoter and enhancer regionsof the thus obtained plasmid pChi641LG11 were converted into MoLTR inthe following manner.

A 3 μg portion of the plasmid pChi641LG11 was dissolved in 30 μl of abuffer solution containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT. The thus prepared solutionwas mixed with 10 units of EcoRI and 10 units of XhoI and incubated at37° C. for 4 hours. The resulting reaction mixture was subjected toagarose gel electrophoresis and about 0.2 μg of a DNA fragment of about8.3 kb was recovered. Next, 0.1 μg of the pChi641LG11 EcoRI-XhoIfragment and 0.1 μg of the pPMOL3 EcoRI-XhoI fragment prepared abovewere dissolved in 20 μl of the T4 ligase buffer. The resulting solutionwas mixed with 175 units of T4 DNA ligase and incubated at 4° C. for 24hours. Using the resulting reaction mixture, transformation of E. coliHB101 was carried out to obtain a plasmid pChi641LGM11 (FIG. 30) as aKM-641 chimera L chain expression vector.

7. Expression of Anti-GD₃ Chimera Antibody in SP2/0 Cells

Introduction of plasmid into SP2/0 cells was carried out making use ofthe electroporation technique in accordance with the method of Miyaji etal. (Cytotechnology, 3, 133–140 (1990)).

The plasmids pChi641LG11 and pChi641HA1 (2 μg for each), or the plasmidspChi641LGM11 and pChi641HAM1 (2 μg for each), were simultaneouslyintroduced into 2×10⁶ of SP2/0 cells, and the resulting cells weresuspended in 40 ml of RPMI1640-FCS(10) which has been prepared bysupplementing RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) with 10%of FCS, 1/40 volume of 7.5% NaHCO₃, 3% of 200 mM L-glutamine solution(available from GIBCO) and 0.5% of a penicillin-streptomycin solution(GIBCO, a solution containing 5,000 units/ml of penicillin and 5,000units/ml of streptomycin). The thus prepared cell suspension wasdistributed in 200 μl-portions into wells of a 96-well microtiter plate(Flow Laboratories), and the cells were cultured at 37° C. in a CO₂incubator. After 24 hours of the culturing, G418 (GIBCO) was added tothe cell suspension to a final concentration of 0.5 mg/ml, and theculturing was continued for 1 to 2 weeks. When transformant colonieswere developed and grown into confluent stages, culture broths wererecovered from the wells to measure anti-GD₃ chimera antibody activitiesby ELISA method in the following manner.

<Enzyme Immunoassay (ELISA)>

A 2 ng portion of GD₃ (available from Iatron) or other type ofganglioside was dissolved in 2 μl of ethanol solution containing 5 ng ofphosphatidylcholine (Sigma Chemical Co.) and 2.5 ng of cholesterol(Sigma Chemical Co.). A 20 μl portion of the thus prepared solution orthe same volume of its dilution solution was distributed into each wellof a 96-well microtiter plate (available from Greiner). Afterair-drying, blocking was effected with PBS containing 1% BSA. To eachwell was added 50 to 100 μl of a culture supernatant of a transformant,a purified mouse monoclonal antibody solution or a purified chimeraantibody solution. After reaction at 4° C. for 10 hours, each well waswashed with PBS and charged with 50 to 100 μl of peroxidase-labeledprotein A (Funakoshi Pharmaceutical Co., Ltd.), followed by 1 to 2 hoursof reaction at room temperature. After washing with PBS, 50 to 100 μl ofABTS substrate solution prepared by dissolving 550 mg of diammonium2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) in 0.1 M citratebuffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxide to the resultingsolution just before its use was added to each well to develop color,and OD₄₁₅ of the reaction mixture was measured.

Among clones thus obtained, culture broth of a clone having the highestactivity measured by ELISA method contained anti-GD₃ chimera antibody inan amount of about 0.1 μg/ml.

The clone having anti-GD₃ chimera antibody activity was suspended in theaforementioned RPMI1640-FCS(10) medium supplemented with 0.5 mg/ml ofG418 and 50 nM of methotrexate (to be referred to as “MTX” hereinafter)to a final cell density of 1–2×10⁵ cells/ml. The thus prepared cellsuspension was distributed in 2-ml portions into wells of a 24 wellplate, and the cells were cultured at 37° C. for 2 to 3 weeks in a CO₂incubator to induce clones resistant to 50 nM MTX. When the thus inducedclones were grown into confluent stages, anti-GD₃ chimera antibodyactivities in the culture broths were measured by the ELISA method.Among clones thus obtained, culture broth of a 50 nM MTX-resistant clonehaving the highest activity measured by ELISA method contained anti-GD₃chimera antibody in an amount of about 0.3 μg/ml.

The 50 nM MTX-resistant clone was suspended in the RPMI1640-FCS(10)medium supplemented with 0.5 mg/ml of G418 and 200 nM of MTX to a finalcell density of 1–2×10³ cells/ml. The thus prepared cell suspension wasdistributed in 2-ml portions into wells of a 24 well plate, and thecells were cultured at 37° C. for 2 to 3 weeks in a CO₂ incubator toinduce clones resistant to 200 nM MTX. When the thus induced clones weregrown into confluent stages, anti-GD₃ chimera antibody activities in theculture broths were measured by the ELISA method. Among clones thusobtained, culture broth of a 200 nM MTX-resistant clone having thehighest activity measured by ELISA method contained anti-GD₃ chimeraantibody in an amount of about 2 μg/ml. This 200 nM MTX-resistant clonewas named transformant KM-871.

Expression of anti-GD₃ chimera antibody protein in the transformantKM-871 was confirmed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) in the following manner.

The transformant KM-871 was suspended in GIT medium (Nihon Seiyaku Co.,Ltd.) supplemented with 0.5 mg/ml of G418 and 200 nM of MTX to a finalcell density of 1–2×10⁵ cells/ml. The thus prepared cell suspension wasdistributed in 100-ml portions in 175 cm² flasks (available fromGreiner), and the cells were cultured at 37° C. for 3 to 5 days in a CO₂incubator. When the cells were grown into confluent stage, the resultingculture broth (about 900 ml) was recovered and subjected to salting-outwith 50% ammonium sulfate. Using Affigel Protein A MAPS-II Kit (Bio-RadLaboratories), about 100 μg of purified anti-GD₃ chimera antibody KM-871was obtained. About 5 μg of the thus purified anti-GD₃ chimera antibodyKM-871 was subjected to electrophoresis in accordance with theconventional method (Laemmli, Nature, 227, 680 (1970)) to check itsmolecular weight. The results are shown in FIG. 31. As shown in thefigure, under reductive conditions, molecular weights of the chimera Hchain and the chimera L chain were found to be about 50 kilodaltons andabout 25 kilodaltons, respectively, thus confirming expression of the Hand L chains having correct molecular weights. Under non-reductiveconditions, molecular weight of the chimera antibody was found to beabout 150 kilodaltons which confirmed expression of the correct sizeantibody consisting of two H chains and two L chains.

8. Reaction Specificity of Anti-GD₃ Chimera Antibody KM-871

Reactivities of the anti-GD₃ chimera antibody with ganglioside GM₁,N-acetyl GM₂ (Boehringer-Mannheim Corp.), N-glycolyl GM₂, N-acetyl GM₃,N-glycolyl GM₃, GD_(1a), GD_(1b) (Iatron), GD₂, GD₃ (Iatron), GT_(1b)(Funakoshi Pharmaceutical Co., Ltd.) and GQ_(1b) (Iatron) were measuredby the ELISA method. In this instance, GM₁ and GD_(1a) were purifiedfrom bovine brain, N-glycolyl GM₂ and N-glycolyl GM₃ from mouse liver,N-acetyl GM₃ from dog erythrocytes and GD₂ from a cultured cell lineIMR32 (ATCC CCL127), in accordance with the conventional method (J.Biol. Chem., 263, 10915 (1988)). The results are shown in Table 1.

TABLE 1 Binding activity of antibody (OD₄₁₅) Anti-GD₃ chimera Mouseanti-GD₃ Ganglioside antibody (0.3 μg/ml) antibody (0.4 μg/ml) N-acetylGM₃ 0.007 0.006 N-glycolyl GM₃ 0 0 N-acetyl GM₂ 0 0 N-glycolyl GM₂ 0 0GM₁ 0 0 GD₂ 0 0 GD₃ 0.717 1.33 GD_(1a) 0 0 GD_(1b) 0 0 GT_(1b) 0 0GQ_(1b) 0 0.16

As shown in Table 1, anti-GD₃ chimera antibody KM-871 and mouse anti-GD₃antibody KM-641 reacted only with GD₃, thus showing no changes in thereaction specificity by the chimera formation.

9. Reactivity of Anti-GD₃ Chimera Antibody KM-871 by FluorescentAntibody Technique

The cultured human malignant melanoma SK-MEL-28 (ATCC HTB72) and G361cells (JCRB) both of which produced ganglioside GD₃ were placed in amicrotube (Treff) to give a cell number of 1×10⁶ cells per tube andwashed by centrifugation (1,200 rpm, 5 minutes) with PBS. 50 μl ofanti-GD₃ chimera antibody KM-871 (10 μg/ml) was added to the microtubeand the mixture was allowed to react at 4° C. for 30 minutes.Thereafter, the cells were washed three times by centrifugation (1,200rpm, 5 minutes) with PBS, then 20 μl of fluorescein isocyanate-labeledProtein A (Boehringer Mannheim-Yamanouchi, 30-fold diluted) was addedand, after stirring, the mixture was allowed to react at 4° C. for 30minutes. Thereafter, the cells were washed three times by centrifugation(1,200 rpm, 5 minutes) with PBS, further suspended in PBS, and submittedfor analysis using FCS-1 flow cell sorter (Nippon Bunko).

Control tests without the addition of KM-871 were performed by the sameanalytical procedure as mentioned above.

The results are shown in FIG. 32. The fluorescence intensity peak forKM-871 showed shifting to the right (increased fluorescence intensity)as compared with the control, indicating that this antibody had reacteddirectly with ganglioside GD₃ on the surface of the SK-MEL-28 and G361cells.

10. In Vitro Antitumor Effect of Anti-GD₃ Chimera Antibody KM-871(Complement-dependent Cytotoxicity:CDC)

(a) Preparation of Target Cells

Suspensions of the target cells, namely SK-MEL-28 cells and G361 cells,in RPMI-1640 medium supplemented with 10% FCS were respectively adjustedto a cell concentration of 1×10⁷ cells/ml, Na₂ ⁵¹CrO₄ was added to aconcentration of 100 μCi/1×10⁷ cells, reaction was performed at 37° C.for 1 hour and, thereafter, the cells were washed three times with themedium. The cells were allowed to stand in the medium at 4° C. for 30minutes for spontaneous dissociation and then centrifuged (1,200 rpm, 5minutes), and the medium was added to adjust the cell concentration to4×10⁶ cells/ml.

(b) Preparation of Complement

Serum from three healthy subjects were mixed to serve as a source ofhuman complement.

(c) CDC Activity Measurement

To U-bottomed 96-well plates was added anti-GD₃ chimera antibody KM-871or anti-GD₃ mouse antibody KM-641 to final concentrations within therange of 0.05 μg/ml to 50 μg/ml. To each well were added 2×10⁵ targetcells. Reaction was performed at room temperature for 1 hour. Thesupernatants were removed by centrifugation (1,200 rpm, 5 minutes), thecomplement solution prepared as described above under (b) was added in150-μl portions (final concentration of 15 v/v %), and reaction wasperformed at 37° C. for 1 hour. After centrifugation (1,200 rpm, 5minutes), the amount of ⁵¹Cr in each supernatant was determined using aγ-counter. The amount of ⁵¹Cr resulting from spontaneous dissociationwas determined by adding to target cells the medium alone in place ofthe antibody and complement solution and determining the amount of ⁵¹Crin the supernatant in the same manner as described above. The totalamount of free ⁵¹Cr was determined by adding 5 N sodium hydroxide inplace of the antibody and complement solution, proceeding as describedabove, and determining the amount of ⁵¹Cr in the supernatant.

The CDC activity was calculated as follows:

${{CDC}\mspace{14mu}{activity}\mspace{14mu}(\%)} = {\frac{\begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{in}} \\{{sample}\mspace{14mu}{supernatant}}\end{matrix} - \begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{resulting}} \\{{from}\mspace{14mu}{spontaneous}} \\{dissociation}\end{matrix}}{\begin{matrix}{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}} \\{{free}\mspace{14mu}{\,^{51}{Cr}}}\end{matrix} - \begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{resulting}} \\{{from}\mspace{14mu}{spontaneous}} \\{dissociation}\end{matrix}} \times 100}$

The results are shown in FIG. 33. It was found from the results thatchimera antibody KM-871 showed strong cytotoxicity against the SK-MEL-28and G361 cells as compared to mouse antibody KM-641, which indicatesthat chimera antibody KM-871 would be clinically more useful than mouseantibody KM-641.

11. In Vitro Antitumor Effect of Anti-GD₃ Chimera Antibody KM-871(Antibody-dependent Cell-mediated Cytotoxicity:ADCC)

(a) Preparation of Target Cells

The target SK-MEL-28 and G361 cells were prepared in the same manner asdesdribed above under 10 (a).

(b) Preparation of Effector Cells

50 ml of human venous blood was collected, 0.5 ml of heparin sodium(Takeda Chemical Industries, 1,000 units/ml) was added, and the mixturewas stirred gently and then centrifuged (1,500 to 1,800 g, 15 minutes)using Polymorphprep (Nycomed Pharma AS). The layer of lymphocytes andpolymorphonuclear leukocytes was separated, and the cells were washedthree times by centrifugation (1,500 to 1,800 g, 15 minutes) withPRMI-1640 medium and suspended in RPMI-1640 medium supplemented with 10%FCS (5×10⁶ cells/ml) for use as effector cells.

(c) ADCC Activity Measurement

To U-bottomed 96-well plates was added anti-GD₃ chimera antibody KM-871or anti-GD₃ mouse antibody KM-641 in 50-μl portions to finalconcentrations of 10 μg/ml. To each well were added 100 μl of targetcells (2×10⁵ cells) and 50 μl of effector cells (5×10⁵ cells) so thatthe ratio of effector cells to target cells should be 50:1 or 100:1.Reaction was performed at 37° C. for 4 hours, followed by centrifugation(1,200 rpm, 5 minutes). The amount of ⁵¹Cr in each supernatant wasdetermined using a γ-counter. The amount of ⁵¹Cr resulting fromspontaneous dissociation was determined by adding to target cells themedium alone in place of the antibody and effector cells and measuringthe amount of ⁵¹Cr in the supernatant in the same manner as describedabove. The total amount of free ⁵¹Cr was determined by adding 5 N sodiumhydroxide in place of the antibody and effector cells, proceeding asdescribed above, and determining the amount of ⁵¹Cr in the supernatant.

The ADCC activity was calculated as follows:

${{ADCC}\mspace{14mu}{activity}\mspace{14mu}(\%)} = {\frac{\begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{in}} \\{{sample}\mspace{14mu}{supernatant}}\end{matrix} - \begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{resulting}} \\{{from}\mspace{14mu}{spontaneous}} \\{dissociation}\end{matrix}}{\begin{matrix}{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}} \\{{free}\mspace{14mu}{\,^{51}{Cr}}}\end{matrix} - \begin{matrix}{{Amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{resulting}} \\{{from}\mspace{14mu}{spontaneous}} \\{dissociation}\end{matrix}} \times 100}$

As a control, the medium was added in place of the antibodies, theprocudure mentioned above was followed, and the amount of ⁵¹Cr in thecontrol supernatant was determined for ADCC activity calculation.

The results are shown in FIG. 34. In both cases of using lymphocytes andpolymorphonuclear leukocytes as effector cells, chimera antibody. KM-871showed strong antibody-dependent cell-mediated cytotoxicity against theG361 cells as compared to mouse antibody KM-641, which indicates thatchimera antibody KM-871 would be clinically more useful than mouseantibody KM-641.

12. In Vivo Therapeutic Effect of Anti-GD₃ Chimera Antibody KM-871(Therapeutic Effect on Transplanted Tumors)

Human malignant melanoma G361 cells (1×10⁷ cells) were intracutaneouslytransplanted to abdominal parts of Balb/c nu/nu mice (5 to 7aminals/group). Anti-GD₃ chimera antibody KM-871 (100 μg/animal) wasintravenously administered into mice four times starting from the nextday of the transplantation of the tumor cells. To the mice of thecontrol group, 100 μg of anti-GD₃ mouse antibody KM-641 or anti-SialylLe^(a) monoclonal antibody AMC-462 (ECACC 86050801) was intravenouslyadministered five times starting from the day of the transplantation.The therapeutic effect on transplanted tumor cells was determined interms of tumor size (volume) calculated by the following equation.Tumor size (mm³)=0.4×a×b ²

-   -   a: major axis    -   b: minor axis

The results are shown in FIG. 35. As shown in FIG. 35, remarkable growthof tumors was observed in the control group to which AMC-462 wasadministered, while the growth of tumors was significantly suppressed inthe group to which KM-641 was administered. KM-871 showed furtherstronger therapeutic effect so that the establishment of tumors wascompletely inhibited 65 days after the transplantation.

REFERENCE EXAMPLE 1

(1) Preparation of Antigen

In 30 ml of chloroform/methanol (2/1) solution were dissolved 5 μg ofganglioside GD₃ having NeuAcα2→8NeuAcα2→3Gal sugar chain on itsnon-reducing end (Iatron), 0.5 μmol of dipalmitoylphosphatidylcholine(Sigma Chemical Co.), 0.5 μmol of cholesterol (Nakalai Tesque), 0.05μmol of dipalmitoylphosphatidylic acid (Sigma Chemical Co.) and 2.5 μgof Lipid A (Funakoshi Pharmaceutical Co., Ltd.). The thus preparedsolution was warmed at 45° C. to remove solvents, thereby obtaining auniform lipid thin film. After completely removing solvents by sackingthe film for 1 hour using a vacuum pump, the resulting film was mixedwith 0.5 ml of PBS solution and stirred at 45° C. to obtain an antigensolution.

(2) Preparation of Antibody-producing Cells

Mice was immunized by administering 0.5 ml of the antigen solutionobtained in the above step (1) into the caudal vein once every week for7 weeks. For further immunization, ganglioside GD₃-positive SK-MEL-28(ATCC HTB 72) cells (1×10⁷ cells) were intraperitoneally administeredonce every week for three weeks. On the third day after the lastadministration, spleen cells were prepared from each mouse for use inthe following cell fusion.

(3) Preparation of Mouse Myeloma Cells

A mouse myeloma cell line P3-U1 having 8-azaguanine resistance wascultured in normal medium (RPMI1640 medium containing 10% fetal calfserum (FCS)) to obtain 2×10⁷ or more cells for use in the following cellfusion as parent cells.

(4) Preparation of Hybridoma

The spleen cells and myeloma cells obtained in the above steps (2) and(3), respectively, were used in ratio of 10:1 and subjected to cellfusion in accordance with the aforementioned procedure. After culturingat 37° C. for 14 days in HAT medium (prepared by supplementing normalmedium with hypoxanthin (10⁻⁴ M), thymidine (1.5×10⁻⁵ M) andaminopterine (4×10⁻⁷ M)) under an atmosphere of 5% CO₂, fused cells wereselected and cultured in HT medium (HAT medium minus aminopterine).Then, active wells were selected by assaying the antibody titers againstganglioside GD₃, and after changing to normal medium, cloning wasrepeated twice. Thereafter, hybridomas which showed specific reactionwith ganglioside GD₃ were selected by enzyme immunoassay orimmunohistological evaluation (ABC method). That is, 2 ng of gangliosideGM₃ (purified from dog erythrocytes in accordance with the method ofNores et al., J. Immunol., 139, 3171 (1987)) and 2 ng of ganglioside GD₃(Iatron) were dissolved in 2 ml of ethanol solution containing 5 ng ofphosphatidylcholine (Sigma Chemical Co.) and 2.5 ng of cholesterol(Sigma Chemical Co.). The thus prepared solution was distributed in20-μl portions into wells of a 96 well microtiter plate (FlowLaboratories), air-dried and then subjected to blocking using 1% BSA-PBSsolution. Each of the resulting hybridoma culture supernatant wasdistributed in 50-μl portions into the plate wells carrying aganglioside GD₃ adsorbed and the plate carrying ganglioside GM₃ adsorbedthereon, and the reaction was allowed to proceed at 4° C. for 18 hours.

After the reaction, a hybridoma strain capable of producing mousemonoclonal antibody specifically reactive with ganglioside GD₃ but notwith ganglioside GM₃ were selected in accordance with the known method(Cancer Res., 46, 4438 (1986)). This mouse monoclonal antibody was named“mouse monoclonal antibody KM-641”, and the hybridoma which producesthis antibody was named “hybridoma KM-641”. The hybridoma KM-641 hasbeen deposited on Sep. 27, 1990, with Fermentation Research Institute,Agency of Industrial Science and Technology, 1-3, Higashi 1 chrome,Tsukuba-shi, Ibaraki, JAPAN, under the Budapest Treaty and has beenassigned the designation as FERM BP-3116.

The present invention provides a process for the production of humanizedchimera antibody wherein the chimera antibody can be produced easilywithout changing any one of amino acids of its mouse antibody variableregion, as well as a humanized chimera antibody specific for gangliosideGD₃.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method of treating cancer that express a ganglioside GD₃ whichcomprises administering to a patient an effective amount of a humanchimeric antibody comprising a heavy chain variable region and a lightchain variable region of a non-human antibody, and a heavy chainconstant region and a light chain constant region of a human antibody,wherein said chimeric antibody binds to the ganglioside GD₃, said heavychain variable region has the amino acid sequence of residues 11 to 129defined in SEQ ID NO: 18, and said light chain variable region has theamino acid sequence of residues 21 to 127 defined in SEQ ID NO:
 19. 2.The method according to claim 1, wherein said cancer is melanoma.